Extracellular matrix protein adherent t cells

ABSTRACT

Substantially purified mature T cells, including αβ T cells and γδ T cells, are capable of binding to an extracellular matrix protein, particularly to one or more of a collagen, a fibronectin, a laminin, a fibrinogen, or a proteoglycan. Also, compositions including the substantially purified ECM binding mature T cells, for use in adoptive immunotherapy in a subject. Also, methods for treating a condition in a mammal, including administering to the mammal an effective quantity of the substantially purified ECM binding mature T cells, and treatment methods using the compositions. Also, methods for increasing the proportion, in a cell population, of substantially purified ECM binding mature T cells. Also, a method for assessing the likelihood that a mixture of cells contains activated T cells capable of localizing to a site in vivo, wherein an extracellular matrix protein is present at the site, in which greater binding of T cells to an extracellular matrix protein on a support in vivo indicates a greater likelihood that a mixture of cells contains activated T cells capable of localizing to the site in vivo.

BACKGROUND OF THE INVENTION

This invention was made in the course of work supported in part by theUnited States Government, and the Government has certain rights in theinvention.

This application is a Continuation-in-Part of my copending applicationU.S. Ser. No. 414,131, filed Sep. 28, 1989, now abandoned, herebyincorporated by reference.

This invention relates to isolation, identification and therapeutic useof populations of T cell lymphocytes, and particularly of populations ofT cell lymphocytes that are capable of binding to specific extracellularmatrix proteins.

The term "leukocyte" refers to any of the nucleated cells normallypresent in blood or tissues whose major function is defense againstforeign invaders of the body. Our understanding today regarding thedifferent types of leukocytes stems from the classic staining researchesof Paul Ehrlich beginning in 1878 whose discovery of polychromatic andsupravital stains led to the first important advances in the knowledgeof leukocytes. His pioneer investigations based on the morphology of thedifferent leukocytes coupled with the first understanding of thematuration sequence via the work of Pappenhein in 1914 led directly tothe classification system used for decades thereafter. Initially, thedifferent types of leukocytes were separated according to: (1) the typeof defense function provided--i.e., phagocytosis, antibody production,or cellular immunity; (2) the shape of the nucleus of the cell--i.e.,polymorphonuclear or mononuclear; (3) the site of origin for thecell--i.e., myeloid or lymphoid; and (4) the presence or absence ofintracellular specific staining granules--i.e., granulocytes ornon-granulocytes. This led directly to the identification andestablishment of normal values of different types of leukocytes asneutrophils, eosinophils, basophils, monocytes (including macrophages),and lymphocytes. Of these five major leukocyte types, the lymphocyte(although first defined as a morphological entity in 1879) has since theearly 1950's been the subject of major, intense, clinical, and researchinvestigations which have led to unparalleled advances in ourunderstanding of how these cells are created, organized, and function invivo.

Lymphocytes are mononuclear cells whose cytoplasm does not contain anyspecific staining granules. As with other leukocytes and red bloodcells, they originate in the bone marrow. Lymphocytes acquire theirimmunocompetence in the bone marrow and thymus, and reside in the organsof the peripheral lymphoid system (lymph nodes, spleen, adenoids andtonsils, the Peyer's patches of the small intestine) where theyencounter antigens and mount immune responses. They also circulate inthe peripheral blood; and may recirculate between the blood stream, thelymphoid organs, and sites of immune reactions in the tissues.

Lymphocytes have been divided broadly into two major orders: B-cellsresponsible for antibody production and the antibody (or humoral) immuneresponse; and T cells responsible for cellular immune responses andimmune regulation generally. In recent years the T cell lymphocyte hasbecome ever more intensively explored.

In general, the present state of knowledge and understanding regarding Tcell lymphocytes is based upon three different and complementaryinvestigational approaches. These are, in summary: (a) study of thefunctional properties of T lymphocyte subsets; (b) study of the surfaceantigens found on T cells and their subsets, using specific monoclonalantibodies; and (c) use of the methods of biochemistry and molecularbiology to investigate mechanisms of T cell functional activities andspecificity. Because of the complexity of the immune system and thenewness and rapidly changing nature of these modern immunologicalstudies (e.g., most of the monoclonal antibodies which have made thesestudies possible were not available before the early 1980's), theresults of these various studies can be difficult to understand andcorrelate. Therefore, the three approaches are discussed in greaterdetail below.

The first investigational basis is the functional designation resultingfrom an empirically observed in vivo and/or in vitro specific biologicalactivity. Such observations provide functional designations including:helper/inducer T cell, cytotoxic/suppressor T cell, lymphokine activatedkiller cell, tumor infiltrating lymphocyte cell, delayedhypersensitivity cell, dendritic epidermal T cell, and intraepitheliallymphocyte (the latter two terms designating T cell subsets that canlocalize in particular areas of the body). Each of these describes anobserved function of lymphocytes. The designations employed are solelyin functional or operative terms because each identification is basedexclusively on the observed capability or attributes of one type of Tcell able to participate in specific biological activities and/orimmunological events.

The second basis of T cell lymphocyte classification resulted from theability to produce specific antibodies which are then employed tocharacterize and define the surface antigenic determinants or cellsurface markers found on different kinds of T cell lymphocytes. With theability to produce monoclonal antibodies such as the OKT series(produced by Ortho Diagnostic System, Inc.), the Leu series (produced byBeckon-Dickinson), and the Coulter series (produced by CoulterImmunology), the individual T cell antigenic designations for man,mouse, and other animals were created. In order to bring some order tothe confusing alternative nomenclatures which have resulted from thedevelopment of various sets of monoclonal antibodies provided bydifferent laboratories, a new nomenclature system was adopted by theFirst International Workshop on Human Leukocyte Differentiation Antigens(Jour. Immunol. 134:659-660 (1985); see also Knapp et al., Immunol.Today 10:253-258 (1989)]. Using this nomenclature system, all monoclonalantibodies that appear to detect a particular antigen are assigned to anumbered "cluster of differentiation" or "CD" for that antigen. It hasbeen found that particular T cell subsets initially defined by function(such as helper and cytotoxic/suppressor T cells) also havecharacteristic cell surface markers as defined by specific monoclonalantibodies. The biochemical function of these cell surface markers isitself an active area of investigation. The phenotyping of the differentT cells and their separation into different subclasses based on theirindividual surface markers has become the most favored investigationaltechnique among researchers today.

The third investigational basis for identifying and distinguishingdifferences among the various populations comprising T cell lymphocytesinvolves molecular biological and molecular genetic studies of proteinsinvolved in immunological activities of T cells, including recognitionand responses to specific antigens. The focus of these studies is the Tcell receptor which is responsible for the recognition of a specificantigen by that individual T cell.

T cell receptors ("TCR") on the classically defined types of cells suchas helper and cytotoxic T cells were found to be composed of twosubunits termed "TCR alpha and beta" proteins. These proteins are thespecific products of individual genes that are themselves rearrangedduring thymic ontogeny (see, e.g., Allison et al., Jour. Immunol.192:2293-2300 (1982); Meuer et al., Jour. Exp. Med. 157:705-719 (1983);Haskins et al., Jour. Exp. Med. 157:1149-1169 (1983)). These TCRalpha/beta ("αβ") protein molecules are found to comodulate, tocoimmunoprecipitate, and to be coexpressed with the CD3 glycoprotein;and the direct physical association of the two protein complexes wasdemonstrated by chemically cross-linking the TCR αβ molecules to the CD3glycoproteins. These αβ T cell receptors do not recognize solubleantigens. Rather, they recognize antigens on cell surfaces, complexedwith proteins of the major histocompatibility complex (or "MHC").Moreover, the MHC protein must be of the correct "self" type (i.e., anMHC protein from the same individual organism or an organism geneticallyidentical at that locus) as the T cell and not from a geneticallydifferent individual. The recognition of such antigens is thus said tobe "MHC restricted". Helper T cells recognize antigens coupled with oneclass ("Class II") of MHC proteins on the surface of antigen-presentingcells such as macrophages; and the surface protein CD4 on the surface ofthese T cells is thought to serve as an accessory binding factor for theMHC protein. Cytotoxic T lymphocytes (or "CTL") recognize the surfaceantigens of their target cells when complexed with another class ("ClassI") of the MHC proteins also found on the surface of the target cell.The surface protein CD8 on the CTL surface again is thought to serve asan accessory binding factor. Recognition of antigen results in a cascadeof events within the T cell which leads to the expression of helper orcytotoxic functions. In the case of cytotoxicity, the CTL lyses itsspecific target cells. (For a general review of these processes, see thetextbook by J. W. Kimball, "Introduction to Immunology," Second Edition,New York, MacMillan Publishing Company, 1986.)

More recently however, there has been an identification of a second Tcell receptor protein complex directly associated and controlled by adifferent third and fourth gene designated gamma/delta (or "γδ") genes.These γδ genes have been identified in the mouse and in man, and cellsbearing γδ TCRs constitute between 1 and 10% of peripheral T cells inboth humans and mice. The present state of knowledge regarding the classof T cells carrying TCRs which are encoded by the γδ genes is documentedby the following representative publications: Brenner et al., Nature322:145-149 (1986); Brenner et al., Nature 325:689-694 (1987); Faure etal., Jour. Immunol. 141:3357-3360 (1988); Janeway et al., Immunol. Today9:73-76 (1988); Bluestone, J. and L. A. Matis, Jour. Immunol.142:1785-1788 (1989); Band et al. Science 238:682-684 (1987); Janeway,C. A., Nature 333:804-806 (1988); and Hercend, T. and R. E. Schmidt,Immunol. Today 9:291-293 (1988).

In short, it is now clear that two lineages of T cells bearing the CD3antigenic complex can be defined based upon the biochemical nature andpresence of the heterodimeric receptor chains expressed as either αβproteins or γδ proteins. The TCR αβ T cells include the major classes ofT cells with classic MHC-restricted helper, cytotoxic, and suppressoractivities.

The function (or functions) of γδ T cells is unknown. Gamma/delta Tcells have been widely demonstrated to carry out non-MHC-restrictedcytotoxic activity against a variety of tumor cells. However, thisrequires high concentrations of interleukin-2; and appears to be alymphokine activated killer (or "LAK") phenomenon, a condition which canbe demonstrated by αβ T cells as well (see, e.g., Matis et al., Nature330:262 (1987); Brooks, Nature 305:155 (1983); Shortman et al., Curr.Topics. Microbiol. Immunol. 116:111 (1986); Maziarz et al., Seventh Int.Congress Immunol., 1989, Abstract). Thus, the non-MHC-restrictedcytotoxicity demonstrated by γδ T cells may not be the usualphysiological function(s) of these cells. Recent studies havedemonstrated that at least some γδ T cells can recognize MHC molecules;and in at least one case, recognize a specific antigen in anMHC-restricted manner (Bluestone et al., Jour. Exp. Med. 168:1899(1988); Kozbor et al., Jour. Exp. Med. 169:1847 (1989)). In anotherlaboratory, γδ T cells have been isolated from the peripheral blood ofpatients with B cell lymphomas that can lyse their specific B celllymphomas in vitro.

A particularly noteworthy feature of γδ T cells is their localization intissues. In the mouse, particular narrow classes of γδ T cells have beenshown to localize in the skin and intestinal epithelia (Asarnow et al.,Cell 55:837 (1988); Goodman and Lefrancois, Nature 333:855 (1988)). Nosuch localization in normal tissues has been found in the human (Groh etal., Jour. Exp. Med. 169:1277 (1989)); however, human γδ T cells doappear to localize in various inflammatory sites. In the case of thehuman gut, although normal levels of γδ T cells have been reported onthe average to be low, they have also been reported to be significantlyhigher (17-33%) in the intestines of children with celiac disease(Russell et al., FASEB Jour. 3:A485 (1989); Spencer and Isaakson(letter) Nature 337:416 (1989)). Gamma/delta T cells have been found innumbers somewhat higher than in blood in the synovial fluid andmembranes of adult rheumatoid arthritis patients (Brennan et al., Jour.Autoimmunity 1:319 (1988)). Moreover, clones of γδ T cells have beenisolated from cerebrospinal fluid of patients with subacute sclerosingpanencephalitis (Ang et al., Jour. Exp. Med. 165:1453-1458 (1987)) andfrom joint fluid of patients with juvenile rheumatoid arthritis (DeMariaet al., Eur. Jour. Immunol. 17:1815-1819 (1987)). In both humans andmice, γδ T cells have been found to localize at anatomically distinctregions of the lymphoid system (Bucy et al., Jour. Immunol. 1412:2200(1988)). In contrast, αβ T cells constitute the bulk of the T cellpopulations in peripheral blood and peripheral lymph nodes, while γδ Tcells comprise at most a few percent. The basis of the ability of γδ Tcells to localize within tissues is at present unknown.

The true biological function and capability of TCR γδ cells remainseffectively unknown as has been explicitly noted in the literature(Bluestone, J. and L. A. Matis, Jour. Immunol. 142:1785-1788 (1989)). Inaddition, although great strides have been made in the last few years bydefining the TCR γδ cells in terms of their tissue distribution andprotein receptor/gene organization format, the true physiological roleand the potential receptor ligand for such TCR γδ cells remains elusive.Although much speculation and hope is placed upon future researchinvestigations for TCR γδ cell populations, there is a continuingignorance regarding methods for isolating such TCR γδ cells withoutusing flow cytometry techniques and apparatus; and a continuing absenceof knowledge or understanding of the attributes or potential uses andapplications for individual populations of TCR γδ cells.

SUMMARY OF THE INVENTION

In general, in one aspect, the invention features substantially purifiedmature T cells capable of binding to an extracellular matrix protein,sometimes referred to herein as ECM binding T cells.

A cell "capable of binding to" an extracellular matrix protein, as thatterm is used herein, is a cell that, when suspended either in a"complex" adhesion medium (a standard culture medium containing serumand (if needed) an additional growth factor (such as, for example, IL-2)and either containing or not containing an extracellular matrixprotein), or in a "simple" adhesion medium (RPMI serum-free cell culturemedium lacking growth factors), and contacted with an extracellularmatrix protein bound on a plastic culture plate surface, adheres to thesurface strongly enough so that the cell is not detached by standardgentle pipetting or washing methods.

"Substantially purified T cells capable of binding to an extracellularmatrix protein", as that term is used herein, means cells derived from asample of T lymphocytes taken from a source animal that has a higherproportion of extracellular matrix protein binding T cells than arefound in the source sample. A mixture of cell types can be"substantially purified" T cells capable of binding to an extracellularmatrix protein, as that term is used herein, provided that cells in themixture are capable in a standard binding assay, of greater binding (interms of proportion of cells bound), reproducibly and to a statisticallysignificant degree, to an extracellular matrix protein than tonon-extracellular matrix proteins, such as BSA or ovalbumin. In suchcell mixtures containing extracellular matrix protein-binding T cellsthat are capable of continuous long term growth, the mixture is"substantially purified" if the extracellular matrix protein-binding Tcells constitute at the least a sufficient proportion of the cells inthe mixture to display, without periodic reactivation, continuous longterm growth through extended numbers of doublings, in serumized media orin media containing a serum substitute and any required growth factorsuch as, for example, IL-2.

In preferred embodiments, the ECM binding mature T cells include γδ Tcells, or αβ T cells. "Gamma/delta" or "γδ" T cells means T cells thatexpress γ and δ T cell receptors ("TCR"); and "alpha/beta" or "αβ" Tcells means ells that express α and β TCR.

In some embodiments, the ECM binding mature T cells are capable ofbinding to a collagen such as, for example, collagen I or collagen IV;to fibronectin; to laminin; to fibrinogen; to a proteoglycan such as,for example, a glycosaminoglycan.

In another aspect, in general, the invention features a composition foruse in adoptive immunotherapy in a subject animal, including thesubstantially purified ECM binding mature T cells in a pharmaceuticallyacceptable carrier for administration to the animal.

"Adoptive immunotherapy", as that term is used herein, means anytreatment of a mammalian subject that entails transfer of living immunesystem cells into the subject. In addition to the T cells certainchemical agents that influence the immune response of the subject by,for example, potentiating the activity or the survival of the cells, orinhibiting immunosuppression, can be administered concurrently to thepatient, and an adoptive immunotherapy composition according to theinvention can include one or more such agents. The immune system cellsfor transfer can be derived from one or more source populations of cellsremoved from one or more animals; and all or a portion of the sourcecells can be removed from the subject being treated, or from anotheranimal. Following removal from the donor or donors, the source cells aretreated before transfer to the subject to change the behavior of thecells and/or to selectively alter the proportions of particular celltypes and/or to expand their numbers.

In another aspect, in general, the invention features a method fortreating a condition in a mammal, including administering to the mammalan effective quantity of the substantially purified ECM binding mature Tcells.

In another aspect, in general, the invention features a method fortreating a condition in a mammal, including administering to the mammalan adoptive immunotherapeutic composition containing ECM binding matureT cells.

In another aspect, in general, the invention features a method forincreasing the proportion, in a cell population, of ECM binding mature Tcells, including steps of providing a cell mixture containing activatedT lymphocytes in a medium, contacting the activated T lymphocytes withan extracellular matrix protein on a support to permit at least aportion of the activated T lymphocytes to adhere to the extracellularmatrix protein on the support, and separating the medium from thesupport together with any cells in the medium not adhering to theextracellular matrix protein on the support.

In preferred embodiments the step of providing a cell mixture containingactivated T lymphocytes in a medium includes steps of contacting a cellmixture containing mononuclear leukocytes including T lymphocytes with asupport, preferably a plastic support surface, to permit monocytes andmacrophages present in the cell mixture to adhere to the support, andseparating the support and any cells adhering to it from the cellmixture comprising T lymphocytes; the step of providing a cell mixturecontaining activated T lymphocytes includes steps of providing a cellmixture containing T lymphocytes and activating cells in the cellmixture; the step of providing a cell mixture including activated Tlymphocytes further includes the step of activating cells in the cellmixture; the cell activating step includes contacting the cell mixturewith a first lymphokine-containing medium; the cell mixture containing Tlymphocytes includes cells derived from a first source animal, and theactivating step includes contacting the cell mixture with lymphocytesderived from a second source animal; the lymphocytes derived from thesecond source animal include irradiated lymphocytes; the activating stepincludes contacting the cell mixture containing T lymphocytes with alectin and with irradiated peripheral blood mononuclear cells; theactivating step includes contacting the cell mixture containing Tlymphocytes with mixed B lymphoblastoid cells (preferablyEBV-transformed) either alone or some combination of irradiatedheterologous peripheral blood lymphocytes and/or a lectin and/orindomethacin; the irradiated peripheral blood mononuclear cells and atleast a portion of the cell mixture containing T lymphocytes are derivedfrom the same source animal; at least a portion of the cell mixturecontaining T lymphocytes is derived from a first source animal, and theirradiated peripheral blood mononucleocytes are derived from a secondsource animal; the activating step includes contacting the cell mixturecontaining T lymphocytes with a mitogenic antibody, preferably amonoclonal antibody, more preferably an anti-CD2, anti-CD3, or an anti-Tcell receptor monoclonal antibody; the contacting step includescontacting the cell mixture containing T lymphocytes with anextracellular matrix protein different in composition from theextracellular matrix protein on the support; the contacting stepincludes contacting the cell mixture containing T lymphocytes with anextracellular matrix protein on a support and to a mitogenic antibody;the extracellular matrix protein on the support is a collagen in any ofits forms, including collagen IV, laminin, heparan sulfateproteoglycans, nidogen/entactin, BM-40/SPARC/bone osteonectin, aglycosaminoglycan, and fibronectin; the extracellular matrix proteindifferent from the extracellular matrix protein on the support is acollagen in any of its forms, including collagen IV, laminin, heparansulfate proteoglycans, nidogen/entactin, BM-40/SPARC/bone osteonectin, aglycosaminoglycan, and fibronectin; the lymphokine in the firstlymphokine-containing medium includes an interleukin, such asinterleukin-2, or interleukin-4; the lymphokine in the firstlymphokine-containing medium is tumor necrosis factor, granulocytemacrophage colony stimulating factor, or γ interferon.

In another aspect, in general, the invention features a method forassessing the likelihood that a mixture of cells contains activated Tcells capable of localizing in vivo to a site at which an extracellularmatrix protein is present, including steps of contacting the mixture ofcells with the extracellular matrix protein on a support, underconditions that permit binding to the extracellular matrix protein on asupport of T cells that are capable of binding to the extracellularmatrix protein, whereby greater binding of T cells from the mixture tothe extracellular matrix protein on the support indicates a greaterlikelihood that the mixture of cells contains activated T cells capableof localizing to the site in vivo.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is concerned with a specific populations of T celllymphocytes; methods for the isolation of these particular populations;and therapeutic methods for using such populations of T cells foradoptive immunotherapy in the treatment of different kinds of cancer. Bythe historical development of this scientific area and via the disparityof investigational techniques and biochemical mechanisms of actionsproposed over the last century concerning lymphocytes in general, theuser must have: a thorough and detailed grounding and understanding ofthe publishing literature particularly over the last fifteen years; anda familiarity and in-depth comprehension of the various investigationalassays, reagents, techniques, and apparatus presently employed toidentify and distinguish among different populations of T celllymphocytes. The complexity, newness, and rapidly changing nature ofmodern immunological investigations have resulted in the failure toprovide precise terms and nonambiguous nomenclature, especially asdifferent laboratories adapt older, existing terminology to fit newobservations. For this reason, a focused and precisely articulatedseries of definitions are provided hereinafter which will serve as thesole and exclusive basis of definition and terminology throughout theentire detailed disclosure of the present invention. In addition, in sofar as is reasonable or plausible, sufficient details regardingcharacteristics, attributes, and capabilities, along with such referencecitations as are appropriate, will be provided whenever possible.

I. Nomenclature and Definitions The CD Cluster Designation of AntigenicCell Surface Markers

The CD cluster designation series has been defined by the FirstInternational Workshop On Human Leukocyte Differentiation Antigens andhas come into general use as the nomenclature to be employed withdetection of cell surface antigens for the classification of mammalianlymphocytes generally. The CD cluster designation is thus synonymouswith any proper designation for the "T series" of surface markers inhuman lymphocytes and the "Ly series", L3T4, etc. of designationstypically employed with murine lymphocytes. In so far as is possible,only the CD cluster designations will be employed herein; and the CDdesignation will be recognized as a proper substitute and replacementfor both the T series and the Ly series, L3T4 etc., as individuallyknown. Accordingly, all mature T cell lymphocytes must be CD3⁺ asdetermined by specific monoclonal antibodies. Similarly, the former "T4⁺" cells will now be designated as CD4⁺ ; and the former "T8⁺ " cellswill now be designated as CD8⁺ cells exclusively. A generalrepresentation of the overlap of CD designations with former terms isprovided by Tables I and II respectively hereinafter.

Lymphokines

These are biologically active substances (such as interleukin-2,gamma-interferon, etc.) secreted by various types of lymphocytes,especially activated T lymphocytes. Other biologically active substancesin the immune system, notably interleukin-1, are secreted by monocytesand macrophages, and are often known as monokines. The distinctionbetween lymphokines and monokines is confusing; for example,interleukin-1 while normally considered a monokine can be secreted bycertain T cells as well as by macrophages (Tartakovsky et al., Jour.Immunol 141:3863 (1988)). For this reason, the term "lymphokine" will beused to refer to both classes of regulatory molecules.

Different types of lymphokines exert their effects on various classes ofimmune cells (monocyte/macrophages, T cells, B-cells) and even on othercell types such as endothelial cells in blood vessel walls, fibroblasts,and, in the case of fever mediated by IL-1, the nervous system.Lymphokine action on immune cells is in part responsible forantigen-specific activation and immune responses. A representativelisting of lymphokines which are either synthesized by or regulate Tcells is provided by Table III below. The usual lymphokine which is usedto support the proliferation of activated T cells in culture (includingTILs), as well as to induce LAK activity in subsets of T cells and NKcells, is IL-2. However, IL-4 has recently been used, either alone or incombination with IL-2, to support the growth of activated T cells and inparticular to support the expansion of TILs from tumor samples (Kawakamiet al., Jour. Exp. Med. 168:2183 (1988); Spits et al., Jour. Immunol139:1142 (1987)). IL-4 when added to human peripheral blood lymphocytes,either alone or in combination with IL-2, suppresses the induction ofLAK activity. However, when added to a LAK culture previouslyestablished in the presence of IL-2, the IL-4 does not affect the cell'scytolytic activity, but does promote the continued growth of the T cells(Spits et al., Jour. Immunol. 141:29 (1988)). As listed in Table III,other lymphokines (notably interleukins-1 and -6) may also beinvolved--especially in combination with other lymphokines and/oraccessory cells in the growth of T cells.

                                      TABLE I                                     __________________________________________________________________________    CLUSTER                   TYPICAL LEUKOCYTE                                                                           LEUKOCYTE                             DESIGNATION                                                                              WORKSHOP ANTIBODIES                                                                          SUBPOPULATIONS                                                                              MALIGNANCIES                          __________________________________________________________________________    CD1 (Thy, p 45,12)                                                                       NA1/34,T6,M241,D47                                                                           Corticothymocytes                                                                           Few T-All and T-LL                    CD2 (T, p 50)                                                                            9.6,Til,35.1   All T-cells forming                                                                         Most T-cell                                      E rosette      E rosette     malignancies                          CD3 (T, p 19-29)                                                                         T3,UCHT1,89bl,38.1                                                                           Mature T-cells                                                                              Most T-CLL & CTCL,                                                            few T-ALL and T-LL                    CD4 (T, p 55)                                                                            T4,Leu3a,91D6  Subset T-cells,                                                                             Few T-All, some                                                 mostly inducers                                                                             T-CLL, all CTCL                       CD5 (T, p 67)                                                                            A50,10.2,Ti,UCHT2,SCI,                                                                       Pan T + subpopulation                                                                       Most T-cell                                      AMG4,T101,Crisl,H65,                                                                         B cells       malignancies,                                    HH9                          some B-CLL                            CD6 (T, p 120)                                                                           12.1,T411,B614,WT31,                                                                         Mature T + subpopulation                                                                    Few T-ALL, most                                  MBG6           B cells       T-CLL & CTCL,                                                                 some B-CLL                            CD7 (T, p 41)                                                                            3AL,4A,CL1.3   Pan T         Most T-All, some                                                              T-CLL & few CTCL                      CD8 (T, p 32-33)                                                                         Leu2a,TB,M236,51.1,                                                                          Subset of T-cells,                                                                          Few T-ALL &                                      UCHT4, 2D2, B9.4.1,                                                                          mostly cytotoxic/                                                                           T-CLL                                            B9.3.1,B9.7.6,B9.2.4,                                                                        suppressor                                                     B9.8.6,B9.11.10,                                                              B9.1.1,C10,T811                                                    CD9 (nT,nB, p 24)                                                                        BA2,DU-ALL-1,FMC8,                                                                           Monocytes     Most non-T-                                      SJ9A-4,WB3                   non-B ALL,                                                                    few B-CLL                             CD10 (nT,nB, p 100)                                                                      J5,BA3,NL-1,24.1,                                                                            Pre-B cell polymorphs                                                                       Most non-T-                                      VIL-AL                       non-B ALL                             CD11 (M,G,u)                                                                             Mol,B2.12,M522 Monocytes and granulocytes,                                                                 Some M4 and                                                     some bone marrow cells                                                                      most M5                                                                       stages of AML some                                                            CML                                   CDw12 (M,G,u)                                                                            20.2,M67       Monocytes and Few M4 and M5                                                   granulocytes  stages of AML                         CDw13 (M,G,u)                                                                            MY7,DU-HL60-4,MCS.2                                                                          Monocytes and Most M1 and                                                     granulocytes  few M4 or M5 stages                                                           of AML some CML                       CDw14 (M,u)                                                                              20.3,5F1,MOP15,M02,                                                                          Monocytes     Few M4 & some                                    MOS1,MY4,MOS30,TM18,         M5 stages of AML                                 MOP9,FMC17                                                         CDw15 (G,u)                                                                              BOH3,B13.9,MCS.1,82H7                                                                        Granulocytes, some                                                                          Most M4 and                                      FMC12,FMC13,WM37,                                                                            bone marrow cells                                                                           some M5                                          DU-HL-60.1,FMC10,WM27,       stages of                                        WM30,G1120,TG8,WM38,         AML, some CML                                    TG1,DU-HL60-3,G2,B4.3,                                                        VIMD5,WV41,IGL0                                                    __________________________________________________________________________

                                      TABLE II                                    __________________________________________________________________________                            PERCENT POSITIVE                                             MOLECULAR                                                                             MONOCLONAL                                                                             THYMO-                                                                              PERIPHERAL                                      ANTIGENS                                                                             WEIGHT* ANTIBODY CYTES T-CELLS COMMENTS                                __________________________________________________________________________    T11 (CD1)                                                                            55,000  OKT 11   95    100     associated with                                        Leu 5                  SRBC rosette receptor                   T10    37,000  OKT 10   95     5      Present on early stem                                                         cells, some B cells,                                                          activated peripheral T-                                                       cells                                   T9     190,000 OKT 9    10     0      Transferrin receptor,                                                         present on activated                                                          T-cells                                 T8 (CD8)                                                                             32,000  OKT 8    80     35     Present on cytotoxic/                          43,000  Leu 2a                 suppressor cells                                       Leu 2b                                                         T6 (CD1)                                                                             44,000  OKT 6    70     0      Equivalent to                                          Leu 6                  murine TL antigen                       T4 (CD4)                                                                             60,000  OKT 4    75     65     Present on helper/                                     Leu 3                  inducer T-cells                         T3 (CD3)                                                                             20,000  OKT 3    20    100     Associated with                                23,000  Leu 4                  the T-cell receptor                            26,000                         for antigen                             T1 (CD5)                                                                             67,000  OKT 1    95    100     Equivalent to                                          Leu 1                  murine Ly-1 antigen                     __________________________________________________________________________

                  TABLE III                                                       ______________________________________                                        LYMPHOKINES    ACTION (partial description)                                   ______________________________________                                        Interleukin-1 (IL-1)                                                                         Involved in activation of resting T                                           cells                                                          Interleukin-2 (IL-2)                                                                         Proliferation of T cells; induction                                           of LAK activity in susceptible                                                natural killer and T cells                                     Interleukin-3 (IL-3)                                                                         Proliferation of mast cells and                                               granulocytes                                                   Interleukin-4 (IL-4)                                                                         Proliferation, activation, and                                                differentiation of B-cells, T                                                 cells, and NKs; inhibits human LAK                                            activity; augments growth of                                                  specific TILs in low-dose IL-2                                 Interleukin-6 (IL-6)                                                                         Growth and differentiation of                                                 B-cells and T cells                                            Tumor necrosis factor                                                                        Cytotoxic for some tumor cells;                                (TNF)          involved in activation of blood                                               vessel endothelial cells                                       Granulocyte macrophage                                                                       Involved in differentiation of                                 colony stimulating                                                                           granulocytes and monocyte/macro-                               factor (GM-CSF)                                                                              phages; stimulates antigen                                                    presenting activity of macrophages                             Gamma interferon                                                                             Stimulates expression of MHC                                                  class II proteins on surface of                                               macrophages, some tumor cells,                                                etc.; stimulates antigen presenting                                           activity of macrophages                                        ______________________________________                                    

T Cell Lymphocytes

T cell precursors originate in the bone marrow; and the vast majority ofT cells mature in the thymus. A key event in this maturation is therearrangement and expression of the TCR gene. Mature T cells are presentin peripheral blood, lymphoid organs other than thymus and bone marrow,in inflammatory sites, and to a lesser extent in tissues. All mature Tcells must demonstrate CD3⁺ determinants and TCR αβ or γδ receptorproteins. Other cell surface antigenic markers (determinants) may or maynot be present. The majority of mature T cells are CD4⁺ CD8-, or CD8⁺CD4-; minority populations of cells which are double negative or doublepositive for CD4 and CD8 also exist.

NK or Natural Killer Cells

All NK or natural killer cells are CD3- and TCR-, i.e., negative forboth αβ and γδ receptor proteins. NK cells often appear as largegranular lymphocytes morphologically; and they commonly express cellsurface markers such as CD16⁺ and Leu19⁺ (NKH-1⁺ ; recently assigned toCD56) in the case of human NKs. Natural killer cells mediate cytolyticactions which do not require the presence or expression of specificClass I or Class II MHC antigens on the target cells and are accordinglydeemed non-MHC restricted per se (Hercend, T. and R. E. Schmidt,Immunology Today 9:291 (1988)). The known target cells which aresensitive to NK cytolytic activity presently represent a limited numberof in vitro established leukemia cell lines, the classic one being K562cells. It should be noted also that NK cells are different anddistinguishable from certain populations of T cell lymphocytes which areCD3⁺. Mature T cells are either αβ receptor or γδ receptor positive; andmay demonstrate, particularly upon activation and culture in mediumcontaining high concentrations of IL-2, a non-MHC restricted cytolyticactivity which resembles the activity of natural killer cells. T cellswhich exhibit such an activity may also express Leu19. The publishedscientific literature has on occasion called such T cells "NK-like"cells or cells having "NK-like activity". These terms will not beemployed at any time herein Rather, only the strict definition andmeaning as stated above for NK cells will be employed.

CTL or Cytotoxic T Lymphocytes

All CTL cells demonstrate MHC-restricted cytotoxicity against targetcells. By definition, therefore, CTL cells all recognize a specificantigen within the context of a specific MHC Class I or Class II antigenon the surface of a target cell before lysis of the cell can occur.Accordingly, the CTL cell is defined by: (a) rearrangement and competenttranscription of genes for the T cell receptor ("TCR") protein, or "Ti"complex, i.e., the Ti genes; (b) expression of the CD3/Ti complex on thecell surface; and (c) cytolytic function that is antigen-specific andMHC restricted (Lanier, L. and J. H. Phillips, Immunology Today 5:132(1986)). This population of cells is different and is to bedistinguished from non-MHC-restricted cytotoxic cells.

Non-MHC Restricted Cytotoxic Lymphocytes

Certain T lymphocytes may express non-MHC restricted cytotoxic activity.Such cells may be αβ or γδ. They include minority populations of T cellsfrom peripheral blood and tissues which display such activity; as wellas other T cell subsets which display this activity when activated andcultured in media containing large concentrations of IL-2. This type ofT cell is defined by: (a) rearrangement of Ti genes; (b) expression ofCD3/Ti on the cell surface; and (c) cytotoxic function that is notrestricted by the MHC antigenic markers. It is noteworthy that this typeof CD3⁺ cytolytic cell has improperly been referred to in the literatureas a "NK" cell because it is capable of lysing natural killer sensitivetarget cells without any requirement for MHC antigen restriction. Thisdesignation and terminology is not correct; these cells should bereferred to as T cells displaying non-MHC restricted cytolytic activity(or, LAK activity, if they display such activity for a wide variety oftumor cells after lymphokine activation) (Lanier, L. and J. H. Philips,Immunology Today 7:132 (1986)). There is evidence that T cells with aknown MHC restricted function may be induced to carry out non-MHCrestricted cytotoxic activity (Matic et al., Nature 330:262 (1987);Brooks, Nature 305:155 (1983); Shortman et al., Curr. To. Microbiol.Immunol. 116:111 (1986); Maziarz et al., 1989, Seventh Int. CongressImmunol., Abstract); and that the TCR determinant of T cells displayingnon-MHC specific activity may not even be involved in that activity(Spits et al., Eur. Jour. Immunol. 15:88 (1985); Phillips et al., Jour.Exp. Med. 166:1579 (1989)). It is therefore possible that T cellsdisplaying non-MHC specific cytotoxic activity may, in addition, haveMHC-restricted, TCR-dependent activities as well. The major differencesbetween classical CTL cells, NK cells, and T cells displaying non-MHCrestricted activity are represented by the information within Table IV.

                  TABLE IV                                                        ______________________________________                                                  MHC         NON-MHC                                                           RESTRICTED  RESTRICTED                                              PROPERTIES                                                                              CTL         CTL         NK CELL                                     ______________________________________                                        CD3/Ti    Yes         Yes         No                                          CD2/E     Yes         Yes         Yes                                         receptor                                                                      CD4       Yes (subset)                                                                              Usually No  No                                                    Class II                                                                      restricted                                                          CD8       Yes         Yes         Yes (subset)                                          Class I                                                                       restricted                                                          Fc (CD16) Usually no  Usually no  Yes                                         receptor/                                                                     ADCC function                                                                 Leu19/NKH-1/                                                                            No          Usually yes Yes                                         (CD56)                                                                        Cytotoxicity                                                                  Inhibited by:                                                                 Anti-CD2  Yes         Yes         No                                          Anti-CD4  Yes (some)  No          No                                          Anti CD8  Yes (some)  No          No                                          Anti-LFA-1                                                                              Yes         Yes         Yes                                         Anti-CD3  Yes         Yes         No                                          Specific  Yes         Yes         No                                          Anti-TCR                                                                      Clonotype                                                                     Antibody                                                                      "LGL"     Yes (some)  Yes         Yes                                         morphology                                                                    Origin    Thymic      Thymic      Bone marrow                                 ______________________________________                                    

TCR Or T Cell Receptor

This is the antigenically and biochemically recognized protein complexon the surface of all mature T cell lymphocytes which is found complexedwith CD3⁺ protein and which determines the specificity of the T cell fora specific antigen The TCR protein complex (or "Ti" complex) typicallybinds or is associated with a specific antigen within the context of aspecific MHC molecule as discussed above. Depending upon whichindividual protein components are associated with the TCR molecule, theindividual cell may be either an alpha/beta (αβ) TCR or a gamma/delta(γδ) TCR protein complex. The vast majority of T cells circulatingwithin normal peripheral blood, lymph nodes, and spleen are αβ T cellreceptor proteins; only about 1-10% of T cell lymphocytes in normalblood circulation are γδ TCR cells. Conversely, there is substantialevidence that the majority of T cells found within at least certainepithelial tissues in the mouse are γδ cells (Janeway, C. A., Nature333:804 (1988)). The TCR protein is also known as the "Ti" complex.

Other T Cell Surface Markers

Some of the other well recognized and functionally associated T cellsurface markers or receptor proteins are those which have now been welldefined by the use of specific monoclonal antibodies. These include theCD4⁺ marker also known as the T4⁺ marker on the human cell which isclassically associated with helper/inducer T cell function. Such CD4⁺cells are believed to be important for accessory binding in that the CD4protein strengthens the binding of the TCR protein to a specific antigenand the MHC Class II on an antigen-presenting cell. In addition, theCD8⁺ (or human T8⁺) marker has been classically associated with bothsuppressor T cells and cytotoxic T lymphocytes in vivo. Such CD8⁺classically exhibit MHC-restricted cytotoxicity in that they recognize aspecific antigen in the context of a specific MHC Class I determinant onthe surface of a target cell with subsequent lysis of the target. Some Tcells have been identified which are lacking both CD4 and CD8 receptormarkers and are therefore said to be "double negative" or "DN" cellsConversely, many populations of tumor infiltrating lymphocytes or "TIL"contain subpopulations which demonstrate both CD4⁺ and CD8⁺ surfacemarkers and are accordingly termed "double positive" T cell lymphocytes.Finally, another major cell surface receptor marker is the Leu19(recently assigned to CD56) determinant found on many kinds of T cellsand NK cells which exhibit non-MHC restricted cytotoxicity.

TIL or Tumor Infiltrating Lymphocytes

These are T cell lymphocytes which are derived from growing resectedhuman or animal solid tumors in culture medium and subsequentlyisolating the lymphocytes from the in vitro cultured tumor tissue. Thisdefinition therefore is exclusively an operational or functionaldefinition by which the obtained lymphocytes are recognized as in vitroobtained cells originating from a solid tumor source alone. When suchsolid tumors are treated by various disassociation methods (Topalian et.al., Jour. Immunol. Methods 102:127 (1987); Kurnick et al., Clin.Immunol. Immunopathol. 38:367 (1986)) and cultured in a lymphokinecontaining media (such as with IL-2), the resulting TIL populationobtained in this manner may be expanded in size and maintained under invitro conditions. Such TIL cells typically are almost entirely T celllymphocytes demonstrating CD3⁺ surface markers; and often demonstratecytolytic activities which can be specific for the individual tumortissue and tumor cells from which they were originally derived. TILs arethus complex mixtures of T cells which can contain various proportionsof cells. (from 0-100%) which are CD4⁺, CD 8⁺, double negative (CD4⁻ andCD8⁻), or double positive (CD4⁺ and CD8⁺). They may also containsubpopulations displaying the Leu19 marker (Rosenberg, S. A., ImmunologyToday 9:58 (1988) and the reference cited therein). Note that productionof TILs requires treatment with a lymphokine such as IL-2; and that TILsobtained in this manner may display specific cytotoxic activity againstthe tumor from which they were derived and/or provided "LAK activity" asdefined below.

LAK or Lymphokine Activated Killer Cells

LAK cells are by definition lymphokine activated (usually usinginterleukin-2) and can be derived by lymphokine induction of either NKcell or T cell types. A LAK cell is thus only an operational/functionaldefinition in which the particular activated cell or cell populationdisplays non-MHC restricted cytotoxic activity for a wider variety ofcell targets and cell types than that usually displayed by NK cells orby non-lymphokine treated T cells displaying non-MHC restrictedcytolytic activity. Such cytolytic activity has been termed "LAKactivity" classically because it is determined empirically by measuringcytolytic activity with an NK-insensitive target cell such as Daudicells. The "LAK phenomenon" results when lymphocytes, either from theperipheral blood or from the solid tissues, are cultured in vitro withlymphokine containing culture media; and thereby acquire the ability tolyse a wider variety of cell types than before treatment with thelymphokine. This operational definition of LAK cells inherentlyrecognizes an empirical cell activation and induction of cytolyticactivity; but does not include or provide any information regardingeither the presence or absence of the TCR protein complex or theindividual CD surface markers originally present (Rosenberg, S. A.,Immunology Today 9:58 (1988); Hercend and Schmidt, Immunology Today9:291 (1988)).

Operationally, the "LAK cells" or "LAK cultures" used in LAK therapy areperipheral blood lymphocytes initially cultured in IL-2 containingmedia, which subsequently display LAK activity. The majority of cellsdisplaying such activity in these cultures are of the NK lineage,although both αβ T cells and γδ T cells with LAK activity are typicallypresent in these LAK cultures.

Extracellular Matrix ("ECM") and Their Components, Including ECMProteins and Basement Membranes and Their Components

Basement membranes comprise a class of extracellular matrix ("ECM")material that is of particular significance for localization bylymphocytes; and basement membrane proteins comprise a class ofextracellular matrix proteins, that play a significant role inlymphocyte localization. Extracellular matrices are insoluble meshworksof proteins and carbohydrates which fill most of the intracellularspaces in the body (Ruoslahti and Pierschbacher, Science 238:492(1987)). Matrices of various types are found in different locations inthe body; and typically are formed of diverse combinations of collagens,proteoglycans, elastin, hyaluronic acid, and various glycoproteinsincluding laminin and fibronectin. Cells of different types interactwith nearly all of the extracellular matrix glycoproteins and collagenswhich have been identified to date. Such interactions can result inadhesion, spreading, migration, growth, and differentiation of cellsinvolved. Basement membranes are the thin extracellular matrices whichsurround epithelial tissues, the outside of endothelial cells incapiliaries and other blood vessels, nerves, fat cells, muscle, andother cell types (Martin and Timpl, Ann. Rev. Cell Biol. 3:57 (1987)).Basement membranes create barriers in vivo which allow embryonic cellsand tissues to segregate and differentiate. They also provide the invivo scaffolding that maintains normal tissue form and serve asmolecular filters in kidney glomeruli and in capillaries to preventpassage of proteins. With respect to immune cells, the basementmembranes constitute barriers to passage of lymphocytes and otherleukocytes out of the bloodstream and into lymph nodes, inflammatorysites, tumors, and other actual or potential sites of immune action inthe tissues of the body. Lymphocytes which are able to target andmigrate towards such sites must therefore possess the capacity totraverse these blood vessel basement membrane barriers at the individualsite of action. With respect to tumor cells, tissue basement membranesconstitute barriers which metastasizing tumor cells must be able totraverse in order to establish metastases. As with other extracellularmatrices, basement membranes are also involved in the adhesion,migration, growth, and differentiation of specific cell types.

The two major components of basement membranes are collagen IV andlaminin. Other components include heparan sulfate proteoglycan, nidogen(also known as entactin), and minor components (which may be variable indifferent basement membranes in the body) including SPARC/boneosteonectin and fibronectin. By definition, therefore, any protein whichcan be isolated from a basement membrane in vivo or in vitro is deemedto be a member of this class of protein. Accordingly, the commoncomponents found in all basement membranes as well as those compositionsfound specifically in basement membranes within particular tissues areall members of this class.

II. Overview Of The Present Invention

It will be recognized and appreciated that presently known methods forthe detection, enumeration, and isolation of γδ T cell lymphocytesdepend upon the use of two individual methods and techniques. These are:(1) the empirical finding that the vast majority of double negativeperipheral blood T cells are presumptively γδ TCR cells based uponbiochemical analysis of the proteins and/or RNA expression, DNArearrangement, and the like (Bank et al., Nature 322:179 (1986); Brenneret al., Nature 322:145 (1986); Rivas et al.. Jour. Immunol. 142:1840(1989)). To identify these cells, the user employs specificfluorescent-labelled monoclonal antibody stains for the CD3, CD4, andCD8 surface proteins; and then utilizes the conventionally knownapparatus and techniques of flow cytometry to isolate the doublenegative cells (Muirhead et al., Bio/Technology 3:337-356 (1985) andreferences cited therein). Those cells which are CD3⁺ but are also CD4⁻and CD8⁻ (double negative) are presumptively believed to be γδ cells.

In the alternative technique, the user employs specific monoclonalantibodies for the TCR protein complex--notably WT31 antibody specificfor the αβ receptor protein or the very recently developed anti-deltareagent, TCRδ1 (Brenner et al., Nature 325:689 (1987) and referencescited therein; Faure et al., Jour. Immunol. 141:3357-3360 (1988) andreferences cited therein). Using this technique, the investigator eitherstains the cells with WT31 antibody and sorts out the cell populationfor WT31⁻ cells; or specifically stains the cells with TCRδ1 antibodyand sorts the stained population for the presence of TCRδ1⁺ T cells. Inthe latter methodology, the apparatus and techniques of flow cytometryare also of maximum value for accuracy and speed of accomplishment; and,indeed, are essential for the separation of cells on the basis of theirlabelling with the fluorescent-labelled antibodies.

This population consists of adhesive γδ T cells, and in the case ofEMATs (described below) represents approximately 5-10% of all peripheralblood γδ T cells and unknown proportions of tissue γδ T cells in thehuman. The activation/adhesion methods do not in all circumstancesresult in γδ T cells; for instance activation of PBL in the presence ofa recall antigen followed by fibronectin binding may yield memory Tcells, which will include mainly helper T CD4⁺ and cytotoxic T CD8⁺TCRαβ cells, as discussed more fully below. And, also as discussed morefully below, I have discovered αβ T cells in the EMAT population, whichare specific adherent T cells.

It will be recognized and appreciated therefore, that the presentinvention is a major departure and deviation from the conventionallyknown methods, techniques, and apparatus for presumptively identifyingand/or isolating presumptive γδ cells and populations. The methodologyof the present invention also provides a number of unique advantages andachievements which were previously unknown and unavailable topractitioners ordinarily skilled in this art. These include:

1. An ability to identify and isolate an individual population of γδreceptor T cell lymphocytes based on their functional characteristicsand properties alone. If the user subsequently wishes to characterizethe isolated population obtained after using the methods of the presentinvention, the user is free to do so using either the conventionallyknown molecular, biochemical, or monoclonal antibody techniques toidentify the various known cell surface markers. As will be notedhereinafter within the experimental data provided, the confirmatoryevidence establishes such cell characteristics for various isolatedpopulations. Nevertheless, such confirmatory procedures are deemedoptional and thus a matter of mere convenience to the individual user.

2. The present methodology does not utilize or require flow cytometryapparatus or techniques whatsoever for the actual isolation andmaintenance of the cell populations.

3. The present methodology provides a means of isolating andcharacterizing γδ T cell subpopulations on the basis of their specificadhesive properties. Current methods are dependent on characterizationand the basis of specific TCR monoclonal antibodies, biochemical andmolecular methods for distinguishing different types of TCR proteins;their location in the body (in the case especially of murine epidermaland gut γδ T cell populations); and, in a few cases, theiridentification based on their reactivity to specific antigens.

Since the physiological functions of γδ T cells are unknown, it is mostdesirable to have this novel means of isolating specific γδ T cellsubpopulations on the basis of this important adhesion property. Theinvention additionally provides a means for characterizing αβsubpopulations on the basis of functional characteristics which areexpected to be related to localization, activation, and function.

A. Obtaining A Mixture Of T Cell Lymphocytes

Initially, one obtains a mixture of T cell lymphocytes as the basis forisolating the population comprising specifically adherent T cells. Forpurposes of the present invention, the source of T cell lymphocytes isneither essential nor critical so long as it is a cell population whichcomprises a proportion of T cells--a percentage of which presumptivelyincludes specifically adherent subpopulations. Accordingly, the sourcemay be: peripheral blood leukocytes taken ex vivo from a living subject;or spleen, lymph node, thymus, or other lymphoid tissue; or a mixedpopulation of leukocytes including lymphocytes which have beenmaintained under in vitro conditions; or tissue samples believed tocontain or supply activatable and/or activated T lymphocytes such astumor tissue as a source of TIL cells; or any other resource of originwherein the mixture of cells includes mononuclear leukocytes of which atleast some demonstrate the presence of the CD3⁺ surface antigen. It isclear therefore that the sole requirements as to source are that somerecognizable T cells as such exist in the material; and a good faithbelief that some proportion of these T cells be specifically adherent Tcells. Conversely, there are no other essential demands or requirementswhatsoever for the cellular mixture comprising the raw material.

It is most desirable, however, that the mixture of cells be prepared toconsist primarily, if not exclusively, of T cell lymphocytes. Forexample, if the source material is fresh peripheral blood taken from aliving subject, it is useful to first isolate mononuclear cells from thewhole blood and Ficoll Hypaque media using conventionally known methods(A. Boyum, Scand. Jour. Clin. Lab. Invest. 21:(Suppl. 97) (1968)). It isalso very desirable to deplete peripheral blood mononuclear cells ofmonocytes and macrophages using conventionally known methods, as forexample, by incubation of the cells in serum containing medium in tissueculture plastic flasks and removing the cells which adhere to theplastic surface (Mosier, Methods in Enzymology 108:294 (1984); Maizel etal., Cellular Immunol. 28:383 (1979)). In this preferred method,peripheral blood mononuclear cells are resuspended (at preferably aconcentration of 10⁶ cells per milliliter) in a suitable lymphocytesupporting culture medium such as LAK medium (comprising RPMI 1640medium containing 10% heat inactivated human serum, penicillin (100units/ml), and streptomycin (100 mg/ml) in plastic T75 culture flasks.By maintaining the mixture of mononuclear leukocytes within the culturemedium in the presence of the plastic solid surface comprising the wallof the T75 culture flask, the vast majority of such monocytes andmacrophages as are present in the cellular mixture become adhered to theplastic solid surface of the flask; while the majority of suchlymphocytes as are present in the mixture remain non-adherent andsuspended within the LAK culture medium contained within the T75 cultureflask. Preferably, the T75 flasks are incubated, flat side down, at 37°C. for about one hour in order to ensure that the monocytes andmacrophages become adherent to the plastic. Subsequently, thenon-adherent cells suspended within the fluid medium are pipetted fromthe flask and centrifuged at about 1,500 rpm for approximately 10minutes in a cell culture centrifuge to concentrate the cells thenremaining. The resulting cell mixture is composed almost entirely of Tcells, NK cells, and B-cells. The B-cells, however, are incapable ofeither growth or activation using the described culture methods and willdie during the first few days of culture.

B. Preparing A LAK Culture From The Lymphocyte Mixture

Once the mixture of lymphocytes has been obtained and preferablyconcentrated, this mixture is then to be activated and prepared as a LAKculture (i.e., a culture of lymphocytes incubated in a medium containingone or more lymphokines such as IL-2 which will result in the inductionof LAK activity). This is preferably performed by suspending the mixtureof T cell lymphocytes (desirably at a concentration of about 2×10⁶ permilliliter) in a lymphocyte supporting medium containing a predeterminedquantity of a lymphokine. Preferably, the culture medium is LAK mediumcontaining 1,000 u/ml of recombinant interleukin-2 (Amgen Company, CetusCompany, or Immunex, Inc.); and culturing the mixture of lymphocytes inthis medium preferably using T75 flasks at 370° C. for approximately24-72 hours. LAK medium is RPMI-1640+10% heat inactivated humanserum+such antibiotics as are deemed necessary, preferably penicillinand streptomycin. Most optimal is an incubation time of approximately 48hours in order that an early stage LAK culture be produced.

It is conventionally recognized that LAK cell cultures may be preparedand produced using a variety of alternate methods. Other lymphokines,especially those which promote the activation and/or growth of T cells,also may be employed. These may be added at the time of or,alternatively, may be added after establishment of the culture. Althoughinterleukin-2 is most preferred for use as a lymphokine of choice, anyconventionally recognized lymphokine (such as those listed within TableIII) which activates and supports the growth of T cells may be employedin varying concentration for this purpose by the user as his needs orconvenience dictates. Moreover, other conventionally known T cellactivation methods (such as the use of lectins, mixed lymphocytereactions, and mitogenic monoclonal antibodies to the CD3 complex or tothe γδ TCR complex), along or in combination with lymphokines producedby recombinant DNA methods or in conditioned media, may also be employedherein for this purpose.

Similarly, the preferred concentration of T cells, the preferredconcentration of lymphokine, and the lymphocyte supporting culturemedium are all matters of personal choice which may be varied greatlyfrom the procedure described herein; and may be altered as desired orrequired to meet specific circumstances and conditions of use.Accordingly, none of these compositions, parameters, concentrations, orchoices of culture medium are controlling or of major importance. Thesole essential requirement and goal is that the mixture of lymphocytesbe maintained in a culture medium containing a measurable quantity of atleast one T cell growth supporting lymphokine which results in theactivation of T cells generally including such specifically adherent γδreceptor T cells as may be present in the mixture.

It will be recognized and appreciated also that if the source oflymphocytes is a tissue sample containing activated and/or lymphokinealtivatable T cells rather than peripheral blood or lymphoid tissue,preparation of activated lymphocytes proceeds in a parallel manner. Forexample, in the case of TIL cells prepared from tumor samples, eitherminced tumor sample or a single-cell preparation from the tumor sample(Topalian et al., Jour. Immunol. Methods 102:127 (1987); Kurnick et al.,Clin. Immunol. Immunopathol. 38:367 (1986)) is incubated in LAK mediumcontaining one or more lymphokine(s)--at least one of which will supportthe growth of T cells and is preferably IL-2.

Early TIL cultures (i.e., actively growing cultures which have expandedto a number of cells which is practical to work with by thesemethods--about 10⁷ -10⁸ lymphocytes) may then be used to isolatespecifically adherent T cells by the earlier described method used forpreparing LAK cultures derived from peripheral blood. However, unlikethe case of peripheral blood lymphocytes, there is no need for a plasticadhesion step to remove macrophage/monocytes (which are few in numberand at least in some ways may be necessary for activation of TIL cells);and the cells resulting from this latter procedure usually contain muchfewer NKs, and in fact are often >90% CD3⁺ T cells.

C. Incubating the Activated Lymphocytes in the Presence of at Least OneExtracellular Matrix Protein

The prepared culture of activated lymphocytes (whether a LAK cultureprepared from peripheral blood or lymphoid tissue; or a TIL culture; oranother preparation of tissue infiltrating lymphocytes) is thensuspended and incubated within a lymphocyte supporting culture mediumcontaining a lymphokine which supports T cell growth and in the presenceof a protein coated solid surface in which the protein coating comprisesat least one basement membrane protein. For purposes of convenience andease, it is most desirable that the internal surface of the multiwellculture plate or the internal wall surfaces of the culture vessel beemployed as the solid support substrate upon which the protein coatingis applied. Alternatively, any other solid material which provides asurface which can be coated with a basement membrane and then submergedin the culture medium may also be used as desired or required. Presumingthe use of a single or multiwell plastic plate as the culture vessel(preferably a non-tissue culture treated plastic petri dish such as aFalcon 1007 dish), it is required that the internal surface be coatedwith at least one extracellular matrix protein as these areconventionally known in composition and structure. Suitableextracellular matrix proteins include but are not limited to all formsof collagen including collagen IV; laminin; heparin sulfateproteoglycans; nidogen/entactin; BM-40 (also known as SPARC or boneosteonectin); glycoaminoglycans; and fibronectin. Alternatively, one mayuse solubilized, multicomponent extracts of whole basement membrane(such as Basement Membrane Matrigel™ sold by Collaborative Research,Inc., which is prepared from the basement membrane of theEngelbreth-Holm-Swarm murine tumor) or crude extracellular matrixpreparations. One would coat the plastic plates with thin gels of thisextract material (i.e., Matrigel) as per manufacturer's direction; andthen dry the gel material overnight on the plate as for the purifiedproteins. The chemical composition and structure of basement membranesare conventionally known and represented by: Martin, G. R., Ann. Rev.Cell Biol. 3:57-85 (1987) and the reference cited therein. Theinteractions of certain tumor cells with basement membranes and theircomponents are also conventionally known. The interactions of nervecells, endothelial cells, and hematopoietic precursor cells in bonemarrow are active areas of research. However, the interaction of Tcells, and in particular γδ T cells with basement membrane components orwith other extracellular matrix components is previously unknown andunappreciated in this art.

Most preferred for use as a protein coating on the solid wall surface ofthe culture vessel is collagen IV (Collaborative Research, Inc.). Thenative murine collagen IV was prepared from the basement membrane of theEngelbreth-Holm-Swarm tumor by the method of Kleinman et al.,(Biochemistry 21:6188 (1982)). Note that only native collagen IV, andnot the denatured form will bind laminin. Preferably collagen IV hasbeen prepared by dissolving the material in sterile 0.1 M acetic acid ata concentration of 100 μg/ml; and then poured onto the internal surfaceof the culture vessel and allowed to dry. In addition, it is desirableto increase the specificity of cell adhesion by taking the preparedcollagen IV (or other basement membrane protein) coated plates andsubsequently treating each collagen IV protein surface with 1% bovineserum albumin to reduce non-specific binding. In the alternative, it isexpected and intended that any of the other conventionally knownbasement membrane or other extracellular matrix proteins may be employedin place of the preferred collagen IV protein in these procedures.

For purposes of the present invention, the prepared activatedlymphocytes may be suspended and incubated in any lymphocyte supportingculture medium which preferably contains a useful concentration of alymphokine which supports T cell growth and viability so long as suchincubation occurs in the presence of the basement membrane proteincoated solid surface (although in particular instances no lymphokine maybe required). However, one or more additional basement membrane proteinsmay be included in the fluid medium with the activated cells;alternatively, these additional proteins may be added as part of theprepared basement membrane protein solid coating or pre-incubated withthat solid protein coating prior to adding the activated lymphocytes.Preferably, one uses plates coated with collagen IV as described aboveand laminin at a concentration of 10 μg/ml mixed in the culture medium.A preferred source of laminin is murine laminin prepared form thebasement membrane of the Engelbreth-Holm-Swarm tumor using the method ofLedbetter et al., (in Methods for Preparation of Media, Supplements andSubstrata for Serum-Free Animal Cell Culture, A. R. Liss, Inc., NewYork, 1984, p. 231), commercially available from Collaborative Research,Inc.

Most desirably, the activated lymphocytes are dispersed at approximately10⁷ cells per plate or well using 3 ml of LAK medium containing 1,000u/ml of interleukin-2, 200 μg/ml of bovine serum albumin and laminin ata concentration of 10 μg/ml. This amount of laminin appears empiricallyto be optimal and desirable. This cellular reaction mixture is thenincubated for 1-12 hours duration, but preferably for only 2-4 hoursincubation, at 37° C. The purpose and result of this incubation intervalis to permit at least a portion of the activated lymphocytes to becomephysically adherent to the solid basement membrane protein coating ofthe support surface.

D. Isolating The Cell Fraction Adhering To The Basement Membrane ProteinCoating

The separation and isolation of the protein coating adherent andnon-adherent cells may be effected using any conventionally known meansso long as there is no substantive damage to that cell fraction adheringto the basement membrane protein coating. Accordingly, one desirabletechnique removes non-adherent cells by washing the plates extensivelywith warm LAK medium initially. Then, using Hank's Balanced SaltSolution ("HBSS") without calcium or magnesium metals but including 1 mMEDTA at 4° C., the protein coating adherent cells become detached fromthe solid protein coating. In this manner, the basement membrane proteincoating adherent cells are detached, collected, and preferablysubsequently concentrated by centrifugation. This isolated,extracellular matrix protein adherent fraction, if derived fromperipheral blood lymphocytes, contains mainly CD3⁻ (presumably NK) cells(between 50%-90%). with the remaining cells being CD3⁺ T cells.Alternatively, if the lymphocytes are derived from TILs, the activatedcells will usually contain fewer NK cells than peripheral bloodlymphocytes; and often the extracellular matrix membrane adherent cellfraction derived from these populations will contain few or no NKs atall. The T cell fraction of the isolated adherent cells was found tocontain between 25-85% γδ T cells, when collagen IV & laminin were used.

E. Reactivating And Culturing The Isolated Cell Fraction

Subsequent to isolation, these extracellular matrix protein adherentcells are subjected to conventionally known reactivation and cultureconditions which supports the growth of T cells. Under these conditions,the NK cells in the extracellular matrix protein binding fraction do notgrow and are lost from the culture. The cells are resuspended inconditioned culture medium (i.e.., filtered medium from the initiallymphokine-containing culture of activated lymphocytes from which theselection with basement membrane protein was done), and subjected toconventionally known T cell reactivation methods. After reactivation,the cells are then resuspended in a culture medium containing alymphokine which supports the growth of T cells, preferably LAK mediumplus between 100-1,000 units/ml of IL-2. The cells, starting with about10⁶ cells recovered from the basement membrane protein selectedfraction, will expand to at least 10¹¹.

The cells may be expanded over time using periodic 1:1 dilutions oflymphokine-containing medium and be maintained at a cell concentrationbetween 5×10⁵ -2.5×10⁶ per milliliter. The culture will then typicallycontain between 15%-90% γδ T cells; and in cultures containing lowerproportions (15%-25%) of γδ T cells, the proportion of γδ T cellsappears to increase to at least 40%-50% with continued growth of theculture.

To aid the user in understanding and using the individual methods of thepresent invention, a detailed protocol for isolating EMATs containingspecifically adherent γδ and αβ T cells is provided. It will beunderstood that the details of the described protocol are thosepresently considered to be most optimal and desirable for use.

Preferred Protocol

1. Coating of Plates

(a) Use Falcon petri dishes (not cell culture treated) 60×15 mm style(Falcon No. 1007).

(b) To each plate, add 3 ml sterile 0.1M acetic acid containing 30 μgcollagen IV. (Collaborative Research provides native collagen IVreceived as 1 mg in 0.05N HCl at 1.32 mg/ml; follow their directions forthawing, etc. Make up solution in 0.1M acetic acid and swirl well.)

(c) Air dry overnight, in the hood, under the UV lamp. Store, wrapped inParafilm or taped to seal, at -20° C.

2. Preparation Of Peripheral Blood Mononuclear Cells (Boyum, A., Jour.Clin. Lab. Invest. 21 (Suppl. 97): (1968))

(a) Draw blood (usually 50 cc) from human volunteer donor, usingheparinized tube. Expect 1-2×10⁶ PBL/ml blood.

(b) Dilute blood 2X with HBSS minus Ca/Mg in 50 ml conical centrifugetubes.

(c) Transfer diluted blood into 15 ml conicals, 10 ml per tube.Carefully underlayer with 3 ml Ficoll-Hypaque, using a long sterileneedle and syringe.

(d) Centrifuge at room temperature, 20 minutes at 2,000 RPM.

(e) Aspirate off most of top liquid with a 5 ml pipette. Pipette up PBLlayer atop Ficoll medium with a plugged sterile pasteur pipette, using acircular motion, being careful not to dip into the granulocyte/red bloodcell layer. Transfer PBL layer to a 50 ml conical and dilute a minimumof 3X with HBSS minus Ca/Mg. (for 50 cc of blood, usually dilute in 100ml HBSS, 50 ml in each of two 50 ml conicals).

(f) Pellet cells by centrifugation at 1,500 RPM, 10 minutes; and decant.Rewash cells one to two more times with HBSS without Ca/Mg to remove theplatelets. Make a cell count before the last centrifugation.

3. Preparation of Macrophage-Depleted LAK Cultures

(a) Prepare Ficoll PBL as described in protocol part 2, above

(b) Remove macrophages (and plastic adherent lymphocytes) by suspendingthe cells at 10⁶ cells/ml in LAK medium (RPMI -1640+10% heat inactivatedhuman serum) and incubate in T75 flasks, ca. 10 ml per flask, treatedside down, at 37° C.

(c) Remove supernatant containing non-adherent lymphocytes, and addsufficient IL-2 to prepare a concentration of 1,000 u/ml IL-2. Incubateat 37° C. in T75 flasks (upright) for LAK culture. Incubate for one ortwo days, etc.

4. Isolation Of Collagen IV Adherent Cells From LAK Cultures

(a) In order to minimize non-specific binding, preincubate collagen IVplates overnight with 3 ml of 1% BSA in HBSS (minus Ca/Mg); remove BSAsolution prior to using the plate.

(b) Count the cells. Centrifuge the cells and save supernatant, afterfiltration through a 0.22 μ filter, at 4° C. or frozen in aliquots, asconditioned medium. Resuspend the pelleted cells at 3.3×10⁶ cells per ml(on the order of 10⁷ cells per plate in 3 ml) in LAK medium+1,000 u/ml.IL-2+200 mg/ml BSA. Put 3 ml of this cell suspension into each plate.

(c) Depending on the experiment, one may then add various amounts oflaminin (from Sigma or Collaborative Research) to each plate. 10 μg/mlappears to be optimal for adherence of cells from LAK cultures, but thismay be greatly varied, especially with respect to populations which mayrequire to be stimulated by addition of laminin for adhesion to collagenIV.

(d) Incubate for various times at 37° C. (ca. 2-4 hours is probablyoptimal).

(e) Wash plates extensively with warm LAK medium to eliminatenon-adherent cells. Adherent cells will appear flat and "blue" under thephase optics of the Nikon Diaphot inverted microscope.

(f) Incubate cells at 4° C. with 3.0 ml per plate of HBSS (minusCa/Mg)+1 mM EDTA for 20 minutes and pipette to remove the cells. Repeatstep if necessary. Remove resuspended cells with a pipette into a 15 mlconical tube and centrifuge cells. Resuspend pellet in 1 ml ofconditioned medium (prepared as described in protocol part 4(b), above);count (using 0.2 ml) the cells and culture the remaining cells in a wellof a plate. Adherent cells are generally between 1-2% of the culture.

5. Reactivate The Cells

Any of a variety of known activation methods can be used, and examplesare outlined in the summary of the invention. The following is presentedas an example.

(a) This step may be achieved by one of two conventionally known methodswhich will reactivate a broad spectrum of T cells, independent of theirantigen specificity:

(i) Reactivation by mixing the cells with an excess (5-10 fold) numberof irradiated peripheral blood mononuclear cells (either autologous orfrom a heterologous donor) together with a T cell stimulatory lectinsuch as phytohemagglutinin (PHA) at a concentration of 1 μg/ml (Williamet al., Jour. Immunol. 133:2986 (1984); Brenner et al., Nature 325:689(1987));

(ii) Reactivation by monoclonal antibodies which are mitogenic for Tcells. Such antibodies are specific either for CD3 (in which case theywill reactivate all classes of mature T cells); or they are specific forvarious classes of TCR proteins themselves, in which case they willreactivate only T cells bearing those classes of T cell receptors forwhich they are specific. In the absence of accessory cells such asmacrophages, these antibodies must be bound to a solid surface, such asthe plastic well of a tissue culture plate, in order to givereactivation of the cells. This is exemplified by the work of Rivas etal. (Jour. Immunol. 142:1840 (1989)) who reactivated γδ T cells usingcell culture wells which had first been coated with goat antimouseimmunoglobulin G (IgG) antibody in order to efficiently bind mouseanti-human CD3 antibodies to the surface of the well; and thensubsequently treated with either of the anti-CD3 antibodies OKT3 (Ortho)or Leu4 (Becton-Dickinson). The cells were then added to the wells inIL-2 containing medium.

Reactivation of the cells (in some cases) may have to be repeated aboutone week later, in order to achieve long-term continuous growth of theculture. Once this has been accomplished, expansion of cultures isolatedfrom 50 cc of blood from normal human volunteers to levels equal to orgreater than 10¹¹ cells have been achieved.

(b) Culture the Reactivated Cells. As cells expand, dilute 1:1 with LAKmedium+100-1,000 u/ml IL-2.

(c) Optional further purification methods for γδ T cells may then beperformed. The above method will yield a growing population containingbetween 15%-90% γδ T cells. Moreover, in populations which have lowernumbers of γδ T cells (ca, 15%-25%) early after reactivation, theproportion of γδ T cells in the culture appears to increase withsubsequent growth to at least 40%-50%. If, especially forinvestigational use, one requires pure specific adherent γδ T cells, orpure specific adherent αβ T cells, conventionally known methodsincluding the following may be employed:

(i) Panning (Wysocki and Sato, Proc. Nat. Acad. Sci. 75:2844 (1978)):This method using anti-TCRδ1 as the specific antibody involvesattachment of the specific antibody to a plastic plate; and uses theantibody to specifically bind those cells which have surface proteinsfor which the antibody is specific. Alternatively, one may use anti WT31antibody (negative selection) to remove T cells. The opposite applies,as will be appreciated, where specific adherent αβ T cells are desired.

(ii) Negative selections via antibody and complement treatment (Cacy etal., Proc. Nat. Acad. Sci. 86:1023 (1989)): Cells are treated with acomplement-fixing antibody against αβ T cells if available; or anon-complement fixing murine monoclonal antibody (such as WT31) followedby a complement-fixing antimurine antibody. This is followed bytreatment with rabbit complement; cells to which the antibody has beenbound will be killed by the complement. Dead cells are then removed bycentrifugation through Ficoll Hypaque. This method may be combined withpanning for more complete purification. The cells may then bereactivated and cultured. If αβ T cells are desired, complement-fixinganti-γδ should be used, as will be appreciated.

(iii) Cloning by limiting dilution (Patel et al., Jour. Immunol.143:1108 (1989)): In wells using irradiated autologous peripheral bloodmononuclear cells in the presence of PHA as feeders (in IL-2 containingmedium), and testing clones for positive staining with anti-TCRδ1 oranti-WT31, respectively. This method will give purified clones of γδ andαβ T cells, the properties of each of which may be investigated. Thismethod thus differs from panning, negative selection withantibody/complement, or use of reactivated and cultured basementmembrane adherent T cells without further purification, since all threeof the later protocols yield bulk, polyclonal populations of cells,which consist largely or entirely of γδ T cells, or of αβ T cells, ifthe appropriate reagents are employed, as will be appreciated.

Cell cloning can also yield purified clones of adhesive (i.e., "EMAT"phenotype, as described more fully below) αβ T cells, which can also beuseful.

V. Characteristics of the Extracellular Matrix Membrane Protein AdherentT Cell Lymphocytes Isolated According to the Invention

The cell fraction able to bind to collagen IV/laminin protein coatingsis about 1-4% of the number of lymphocytes present in two day old LAKcultures prepared from normal human peripheral blood. The binding ofthese cells to an extracellular matrix protein appears to take place infour hours or less under the conditions of the preferred protocol.Similarly, the binding to collagen IV/laminin was found to be betweenabout 60-85% specific as compared to non-specific binding to untreatedplastic plates with cells obtained from LAK prepared cultures. Ingeneral, the isolated adherent cell fraction, if derived from peripheralblood lymphocytes, contains mainly CD3⁻ (presumably NK) cells (between50%-90%), with the remaining cells being CD3⁺ T cells. In the case ofCollagen IV/laminin adherent cells derived from peripheral blood, the Tcell fraction contains between 25%-85% γδ T cells. After reactivationand culture, the growing cell population has been found to contain over90% T cells, of which between 15%-90% are γδ T cells. Both αβ and γδ Tcells are at least largely specifically adherent, as described below. Incomparison, the binding to collagen Iv/laminin of activated lymphocytesderived from other sources, including TILs, is highly variable. TILsusually contain fewer NK cells than peripheral blood lymphocytes (andoften contain little or no NKs), so the basement membrane adherent cellfraction derived from these populations will contain fewer or no NKs).

Within the isolated and cultured T cell population, the cells wereeither CD4⁻ CD8⁻ (double negative) cells, or CD4⁻ CD8⁺. When one ofthese populations was tested with the anti TCRδ1 antibody which isspecific for a common epitope of the delta receptor protein in the γδ Tcell receptor, 80% were found to stain delta (δ) positive. It is clearthat the majority of the double negative and the CD8⁺ T cells in thisisolated population bear γδ receptor proteins. In addition, these cellswere found to be cytotoxic for both K562 cells (a NK-sensitive tumorcell line) and Daudi cells (a NK-resistant tumor cell line).

To further empirically document and confirm these observedcharacteristics, a series of individual experiments was performed as aredescribed hereinafter. The purpose of this empirical description anddata is merely to further demonstrate the representative membership ofthe T cell lymphocyte population isolated and cultured collectively bythe methods of the present invention. It will be expressly understoodand recognized that the experiments and/or empirical data are merelyrepresentative of the present invention as a whole; and do not in anymanner serve to limit or restrict the as present invention to either theexperimental design or the empirical results presented.

VI. Useful Properties of Basement Membrane Protein Adherent γδ T CellsIsolated According to the Invention and their Therapeutic ApplicationsProperties

1. Gamma/delta T cells, which constitute a minority of T cells inperipheral blood and lymphoid organs, appear to demonstrate localizationin tissues. In the mouse, particular narrow classes of γδ T cells havebeen shown to localize in the skin and intestinal epithelia. Although nosuch localization in normal tissues has been found in the human, humanγδ T cells appear to localize in various inflammatory sites in thetissues. Gamma/delta T cells are reported to localize around highepithelial venules of lymph nodes; these have the following attributesof blood EMATs: they are associated with basement membrane, they have anamoeboid morphology, and they belong to the δTCS1⁻ subgroup of γδ TCR.

2. Populations of γδ T cells isolated by the preferred protocol andactivated by IL-2 can bind to collagen IV in the presence of laminin.These cells are extracellullar matrix adherent T cells and are thuscalled "EMAT" cells. Collagen IV and laminin are the major components ofbasement membranes which provide support, boundaries, and oftenessential growth and differentiation stimulation for capillaryendothelial cells and epithelial cells in most tissues in the livingbody. Capillary endothelial cells and their associated basementmembranes, as well as basement membranes associated with other tissuecomponents, also constitute in vivo barriers to penetration of T cells,other immune cells such as B-cells, macrophages, and granulocytes, andof tumor cells. Many cells which bind to basement membranes can alsopenetrate these barriers in vivo. For studies on relationships betweenbinding to basement membrane components and invasion of tissues bymetastatic tumor cells, see R. H. Kramer et al., Int. Jour. Cancer26:639 (1980); and V. P. Terranova et al., PNAS 80:444 (1983).

Activated EMATs bind to basement membranes. The penetration ofendothelial cells of capillaries and venules, and especially of highendothelial venules, may be restricted to capillaries either in areas ofinflammation (including tumors); damaged blood vessels (i.e., bloodvessels which display naked basement membranes--including deranged bloodvessels in many solid tumors); or capillaries and venules in areas ofthe body for which the EMAT cells have specific homing receptors. Seefor example, A. Duijvestijn and A. Hamann, Immunology Today 10:23(1989); Yednock and Rosen, Adv. Immunol. 44:313 (1989); and Stoolman,Cell 56:907 (1989) for reviews of lymphocyte homing, recirculation, andknown and postulated roles for adhesion molecules in tissue andorgan-specific homing of lymphocytes. Note also that special classes ofmurine γδ T cells which are found in specific tissues appear to homespecifically to those tissues, although the precise mechanisms for thishoming phenomenon or any relationship for the role of adhesion moleculesin tissue-specific homing of lymphocytes is as yet unknown. In additionto cells in the peripheral blood, there are γδ T cells in epithelialtissues, in inflammatory sites, and (as demonstrated herein) among tumorinfiltrating lymphocytes in vitro. The latter, under the proper cultureconditions and isolation methods, can be isolated from in vitro culturedTILs.

3. Almost all activated γδ T cells which have been empirically assayeddemonstrate cytotoxicity for allogeneic and in at least some cases,syngeneic tumor cells; and in one case the cells are highly specific forB cell lymphomas, namely specific for an idiotypic determinant ofsurface immunoglobulin. The syngeneic case is one in which doublenegative (and therefore presumably γδ T cells from the peripheral bloodof melanoma patients have also been found among T cell mixtures whichbecome activated when exposed to melanoma cells from the same patient(P. Hersey et al., Cancer Immunol. Immunother. 22:15 (1986)). Cytotoxicactivity of γδ T cells includes non-specific LAK-like activity; and, atleast in some cases, cytotoxic activity which may be specific forcertain MHC proteins (Bluestone and Matis, Jour. Immunol. 14:21785(1989)). LAK-like cytotoxic activity for EMAT cells has been empiricallydemonstrated.

Extracellular Matrix Adherent T Cell Populations Enriched for TCR γδCells

Receptors that mediate interactions of cells with extracellular matrix(ECM) proteins are of key importance in localization and function ofmany types of cells (see, Ruoslahti and Pierschbacher, Science 238:491-497, 1987; Hynes, Cell 48: 549-554, 1987; M. E. Hemler, Annu. Rev.Immunol. 8: 365-400, 1990). Recent work indicates that this is true forcells of the immune system. Savagner et al., Jour. Cell Biol. 103:2715-2727, 1986, describes blocking of migration of avian precursorcells into the embryonic thymus by antibodies against fibronectin,laminin, and the β₁ chain which is common to the "very late antigen"("VLA") subfamily of integrin adhesion receptor molecules, and also bythe peptide arginine-glycine-aspartic acid-serine ("RGDS"). Thisindicates that this migration is in part dependent upon integrins whichare receptors for the ECM proteins fibronectin and/or laminin, and whichrecognize RGD sites on these proteins. ECM interactions may also beimportant in later development of T cells within the thymus; Cardarelliet al., PNAS 83: 2647, 1986, and Cardarelli et al., 1988, Jour. CellBiol., 106:2183 describe adherence to fibronectin by about 10% of murinethymocytes, largely of CD4⁻ CD8⁻ and CD4⁺ CD8⁺ immature phenotypes.

Receptors for ECM proteins have also been implicated in migration andfunction of mature T cells. In particular, the integrin VLA-4, describedby Wayner et al., J. Cell Biol. 109: 1321-1330, 1989, and Guan et al.,Cell 60: 53-61, 1990, to be a receptor for a non-RGD peptide sequence inthe heparin binding CS-1 domain of fibronectin, is described by Eliceset al., Cell 60: 577-584, 1990 to be a receptor for a distinct molecule,vascular cell adhesion molecule-1 (VCAM-1). VCAM-1 is expressed on thesurface of cytokine-activated human endothelial cells, and may mediateadhesion and entry of lymphocytes into extravascular tissues at site ofinflammation (see, Osborn et al., Cell 59: 1203-1211). Holzmann et al.,Cell 56: 37-46, 1989, and Holzmann et al., EMBO Jour. 8: 1735-1741, 1989identify a molecule which appears to be the murine analogue of VLA-4 anda variant form of murine VLA-4 known as VLA-4P as homing receptors whichmediate adhesion to high endothelial venules (HEV) in Peyer's patchesand entry into the mucosal lymphoid organs in the intestine. Inaddition, the non-integrin receptor CD44 has been implicated by Jalkanenet al., J. Cell Biol. 105: 983-990, 1987, and Haynes et al., Immunol.Today 10: 423-428, 1989 as one of the receptors involved in homing toperipheral lymph nodes and mucosal lymphoid organs via high endothelialvenules (HEV) in the mouse and the human, and by Carter et al., J. Biol.Chem. 263: 4193-4201, 1988 in adhesion to the ECM proteins collagen andfibronectin. Thus the same proteins on the surface of T cells which arereceptors for ECM may also be receptors for cell surface molecules onendothelial cells which mediate localization of the T cells in lymphnodes, mucosal lymphoid organs, and in inflammatory sites.

Mature T cells which bear the TCR γδ have been found to exhibit specificlocalization and recirculation properties. In the mouse, TCR γδ cellswith distinct TCR repertoires have been found to localize respectivelyin the skin (Asarnow et al., Nature 341: 60-62, 1989, and Asarnow etal., Cell 55: 837-847, 1988), in the intestinal epithelium (Takagaki etal., Nature 339: 712-714, 1989, and Asarnow et al., 1989), and inepithelia of the vagina, uterus, and tongue (Itohara et al., Nature 343:754, 1990). TCR γδ cells appear to constitute the majority of T cells inthese murine epithelia. Moreover, Mackay et al., Jour. Exp. Med. 171:801-817, 1990 found that a large fraction of peripheral sheep TCR γδcells preferentially follow the same recirculation pathway (blood totissues to afferent lymph) as memory T cells. This is different from therecirculation pathway (blood to lymph nodes via high endothelial venules("HEV") (see, Butcher, Curr. Top. Microbiol. Immunol., 128: 85-122,1986, and Duijvestijn et al., Immunol Today 10: 23- 28, 1989) which isfollowed by the majority of peripheral blood T cells, which are of naivephenotype.

In humans, however, no exclusive or majority populations of TCR γδ cellshave been seen in association with normal epithelial tissue. Neither TCRαβ or TCR γδ cells have been found to localize in normal epidermis, andTCR γδ cells have been found to constitute a small minority of T cellsin the normal gut (see, Groh et al., Jour. Exp. Med. 169: 1277-1279,1989; Bucy et al., Jour. Immunol. 142: 3045-3049, 1989). However, themajority of human gut TCR γδ cells appear to preferentially localize inthe intestinal epithelial layer (Bucy et al., 1989). Moreover, TCR γδcells in human tonsils and peripheral lymph nodes have been frequentlyseen beneath the epithelia of these organs, and in association withsmall blood vessels, at least many of which are HEV (see, Fallini etal., Jour. Immunol. 143: 2480-2488, 1989). Therefore, as in the case ofthe mouse, a fraction of TCR γδ cells appear to localize to structures(epithelial cell layers and small blood vessels) characterized bybasement membranes; TCR γδ cells in these locations are indeedfrequently seen in association with the basement membranes themselves.However, it is not known whether TCR γδ cells which localize in aparticular human organ or tissue have a distinctive, tissue-specific TCRrepertoire. The difference between the mouse and the human with respectto TCR γδ cell localization could either be due to differences inmigration patterns in TCR γδ cells between the two species, ordifferences in migration patterns of subsets of TCR αβ cells whichspecifically localize in epithelial tissues.

The mechanism by which TCR γδ may home to specific tissue sites isunknown. As has been discussed by Itohara et al. (1990), it is unlikelythat localization of a particular subset of TCR γδ cells is directlydetermined by its TCR, since in transgenic mice one can obtainlocalization in a particular tissue of cells expressing TCR γδ geneswhich are different from TCR γδ genes normally expressed in T cellsfound in that tissue (see also, Ferrick et al. Cell 57: 483-492, 1989).An alternative explanation is that specifically homing TCR γδ cells ofdistinct lineages differ not only with respect to their TCR, but alsowith respect to homing receptors and perhaps other properties.

I have identified and substantially purified from the peripheral bloodof normal human subjects and from an epithelial thymus tumor humanlymphocytes that bind to collagen IV in the presence of laminin. T cellsin these populations are enriched for TCR γδ cells, and can be expandedto high cell numbers in IL-2 containing media. The cultured T cellsexhibit specific adhesion to collagens I and IV and to fibronectin. Thehigh proportion of TCR γδ cells in these extracellular matrix adherent Tcell ("EMAT") populations suggests that these cells may constitutesubsets of cells with distinctive adhesion properties, which can beinvolved in localization in specific tissue sites and/or specifictrafficking during immune responses. Both γδ T cells and αβ T cells inthis EMAT preparation exhibit specific adhesion properties, namelybinding to collagen>fibronectin and not laminin or fibrinogen, and canexhibit localization properties.

Phenotypes of Cell Lines Cultured From Collagen/Laminin AdherentLymphocytes

Cell surface markers present on cells of two cell lines cultured fromcollagen/laminin adherent IL-2 cultured PBL (PW-EMAT and ZB-EMAT) and oncells of the cell line cultured from the collagen/laminin adherent TILs(AKT-EMAT) were determined. Although the initial adherent subpopulationsof IL-2 cultured PBL may contain a majority of CD3⁻ cells, at least thevast majority (≧95%) of cells in the cell lines derived from theseinitial populations are CD3+ TCR+ T cells. T cells thus appear to have astrong selective advantage in the activation and culture proceduresemployed in the expansion of these cell lines. As in the case of theinitial adherent populations, the cultures derived from them are highlyenriched for TCR γδ cells. The ratio of αβ to γδ cells, however, isvariable with growth and activation conditions, as has been seen withother mixed populations which contain both αβ and γδ T cells. Thisvariability is seen even with the same cell line at different times whenfrozen samples are thawed, reactivated, and expanded. The culturesusually contain little or no CD4+ cells. However, as shown in FIG. 2,the cultures (especially AKT-EMAT, which is the one TIL derived EMAT)can at times (although rarely) exhibit significant numbers of CD4+cells, which, however, appear to express low amounts of CD4.

Double staining for CD8 and the TCR reagents WT31 and TCR γδdemonstrated that at least the great majority of αβ T cells were CD8⁺and that γδ T cells included both CD8⁺ and CD4⁻ and CD8⁻ (doublenegative) phenotypes.

Adhesion to Extracellular Matrix Proteins by T Cells in EMAT Lines

Each of the three EMAT lines tested (PW-EMAT, ZB-EMAT, and AKT-EMAT)showed specific binding to collagen IV, collagen I, and fibronectin, butbinding to laminin and fibrinogen was no greater than binding to thecontrol proteins BSA and ovalbumin. Adhesion was approximately equalbetween collagens IV and I, and greater to the collagens than tofibronectin by a factor of about two.

Since each of the three cell lines are cell mixtures containing both TCRαβ T cells and γδ T cells, it was important to determine which of thesetwo subsets included cells which were adherent to the extracellularmatrix proteins. Cells of each of these EMAT cell lines (twoPBL-derived, one TIL) which bound both to collagen IV and to fibronectinwas found to contain both TCR γδ and TCR αβ fractions. This indicatesthat both TCRγδ cells and TCR αβ cells in the EMAT populationdemonstrate specific adhesivity, and that the αβ and γδ subpopulationsbind similarly with respect to different extracellular matrix proteins.

Morphology of Cells in EMAT Populations

EMAT populations include cells which exhibit abundant and rapidextension of filopodia, ameboid motion, and adhesion and spreading oncell culture plates. Cells in EMAT populations which adhere to collagensIV and I or to fibronectin also exhibit cytoplasmic spreading, abundantfilopodia (in some cases assuming a "neuron-like" form with axon-likecytoplasmic extensions), and rapid, ameboid changes in cell shape.

Method of Isolation

The preferred method of isolation of EMATs involves use of a complexmedium, including serum, in addition the use of both collagen IV (boundto the plate) and laminin (in the medium). It is designed to provide anyserum factors that may be needed for cell spreading, and is analogous inthis respect to methods for isolating minority subpopulations ofplastic-adherent cells from IL-2 activated PBL.

I have demonstrated isolation of EMAT from all normal peripheral bloodsamples tested (6 donors), and from one TIL. However, the majority ofneither αβ nor γδ T cells from either the IL-2 activated PBL culturesbind to collagen IV/laminin plates. Moreover, I observed no adhesion tocollagen IV/laminin plates in several T cell populations, all of whichcontain γδ T cells at least as a minority: 1. TIL derived from thesecond metastasis from the same patient from which AKT-EMAT wasisolated. 2. a TIL population obtained from Kurnick's lab, which hasbeen subjected to long term expansion, including several reactivationswith feeder cells and PHA.

The adhesive "EMAT" phenotype may be seen in cells of various TCR types,including: 1) αβ (diversity of v.sub.β and v.sub.α use unknown); 2) γδTiγA⁺ δTCS⁻ (as in PBL-derived EMAT); 3) γδ δTCS⁺ TiγA.sup.± (as in EMATfrom AK-TIL). "EMAT" is here taken in a strict sense to refer to cellshaving the binding pattern collagen IV>collagen I>fibronectin, and notlaminin or fibrinogen.

In humans, unlike the mouse, no exclusively γδ T cell population is seenat any site in normal epithelial tissues. This may be owing to a greaternumber in the human of αβ cells, possibly including αβ EMATs, havingspecific epithelial homing. Even in the mouse, a presence in the gutepithelial mucosa of both αβ and γδ T cells having a specific phenotype,common to αβ and γδ subsets, has been described. The mechanism (ormechanisms) of localization of these cells, and whether the mechanism isrelated to ECM and/or other receptors, is unknown.

The EMAT phenotype, as determined by adhesion assay without serum, asdescribed above, includes: unusual, high collagen binding (both collagenI and IV about equal); fibronectin binding; and no specific binding tolaminin or fibrinogen.

The nature of receptors on EMATs is unknown. RGD experiments suggestintegrins, at least in part. The integrin collagen receptors VLA-1 andVLA-2, commonly found on long-term activated T cells, are not inhibitedby RGD peptides. VLA-3 (which may bind to collagen, fibronectin, andlaminin) and VLA-5 are inhibited by RGD peptides, and are candidates forreceptors involved in the adhesion observed in EMATs, however. New,unknown receptors may also be present.

Morphological Aspects of EMATs

EMATs include T cells that exhibit a high degree of motility andpseudopod formation. In adhesion assays, at least a high proportion ofEMATs not only adhere, but also spread and form processes on bothcollagens and fibronectin. In preliminary panning experiments with TCRδ1antibody, motile cells in the population include both antibody binding(presumptively γδ) and non-binding (presumptively αβ) cells.

EMATs may represent specific lineages or they may represent a functionalcategory. Memory T cells represent a functional category of cells whichexhibit special adhesive properties and integrin-mediatedco-stimulation, and also appear to have specific homing properties, atleast some of which may be mediated by their receptors for extracellularmatrix. Memory T cells are co-stimulated via fibronectin (VLA-5) andcollagen, but, unlike in EMATs adhesion, fibronectin co-stimulation inmemory T cells is about two to five times that of collagen.

Adhesive properties of EMATs may reflect a presence of receptors whichare involved in homing and localization (as suggested by the highproportion of γδ T cells among the EMAT subset), and/or a presence ofreceptors which are involved in trafficking and localization of alreadyactivated cells (as are the cells actually observed here) at a site ofaction.

Note that for adhesion to collagen IV, at least under the conditionsused herein, most T cells clearly do not bind, even long term activatedpopulations (such as for example Kurnick TIL) which one would expectexpress abundant VLA-1 and VLA-2. At least many PBL T cells, andespecially memory T cells, have been reported to bind to fibronectinwhen acutely activated, i.e., they are said to be transiently adhesive;this is also reported to be true for CD4⁺ cells on laminin. EMATs areadhesive during a substantially more prolonged period.

Certain transformed T cell lines (i.e., not normal, having lost controlof growth and displaying long term continuous growth without need foractivation) have been reported to bind to fibronectin. In addition,certain normal murine dendritic epidermal T cell ("DETC") lines whichdisplay long term continuous growth under appropriate conditions, whichare γδ, have been reported to bind fibronectin. However, these murinecells exhibit a different adhesion pattern from that of EMATs (DETCsbind to fibrinogen>fibronectin>vitronectin, and not to collagen).Prolonged adhesivity to ECM substrates may be related to long termactivation. Alternatively, however, maintenance by normal T cells ofactive adhesivity, via an unknown mechanism, may be a factor inmaintaining activation, as binding of ECM substrates by helper T cellshas been described as giving an activating signal to the cell. Althoughadhesivity of ECM receptors may be related to long term continuousgrowth, the spectrum of ECM substrates to which the cell binds(different for human EMATs and murine DETCs) may be related to whichreceptors are both expressed and activated.

Extracellular matrix adhesion may be related to expression of receptors,and/or to activation of receptors. But expression alone of receptors isnot sufficient for adhesion, as shown by expression of VLA-5 on restingT cells but no fibronectin binding.

"Activation" may involve linkage of the receptors to the cytoskeleton.Such linkage can be involved both in molecular signalling (i.e.,activation) and in adhesion and spreading on the extracellular matrixprotein layer. As with some other non-T cell systems, the presence ofECM receptors on EMATs may be involved in organizing the cytoskeleton,as differences have been observed in organization of the cytoskeleton indifferent subsets of γδ T cells; EMATs are adhesive and also have activecytoskeletal activity, both characteristics necessary for spreading on asubstrate.

Adhesive T cell types (i.e., EMATs, DETCs, helper T cells) could thusdiffer from other normal T cells with respect both to expression ofreceptors (quantitatively and qualitatively) and other components whichaffect "activation" of the receptors.

In addition to localization, extracellular matrix receptors on T cellsmay also be mediators of T cell activation. Matsuyama et al. (J.Experimental Medicine 170: 1133-1148, 1989) demonstrated synergy betweenanti-CD3 antibody and fibronectin (and to a lesser extent, collagen) instimulating proliferation of resting CD4⁺ murine PBL. This effect offibronectin could be blocked by antibodies to CD29 (the β₁ integrinchain) and by specific antibodies to VLA-5, and by an RGD-containingpeptide. Conversely, stimulation of the CD3/TCR complex has beenreported to affect the adhesive activity of cell surface integrins.Shimizu et al. (Abstract. FASEB J. : A1018, 1990) reported that acuteactivation of resting CD4⁺ human PBL by either crosslinking of theCD3/TCR complex with anti-CD3 or the phorbol ester PMA resulted instrong adhesion of the cells to fibronectin and laminin. Studies withspecific antibodies and inhibitor peptides were reported to indicatethat adhesion to laminin was mediated by VLA-6, adhesion to fibronectinvia its central RGDS (arginine-glycine-aspartate-serine) binding sitewas mediated by VLA- 5, and adhesion to the CS-1 domain of fibronectinby VLA-4. CD3⁺ human PBL acutely activated with TPA, Con A plus TPA, orCon A plus accessory cells have also been reported to demonstrateadhesion and spreading on fibronectin (Kurki et al., Scand. J. Immunol.26: 645-652, 1987); unstimulated cells showed less binding and littlespreading. Adhesion in this study was inhibited 90% by RGDS peptide andbinding was also seen to a proteolytic fragment of fibronectincontaining the RGDS cell binding site, so it was likely to be at leastlargely due to VLA-5. T lymphocytes may express VLA proteins but arereported to demonstrate little or no adhesion to their ECM proteinligands. For example, fresh peripheral blood T cells were found toexpress substantial amounts of VLA-4, VLA-5, and VLA-6, yet demonstratelittle binding to fibronectin or laminin. Treatment of these cells withmitogens, as described above, may affect the adhesive activity ofalready expressed adhesion receptors in addition to effects onexpression.

One case in which expression and activity of adhesion receptors iscorrelated with altered T cell localization and function is that ofmemory T cells. Peripheral blood T cells which exhibit strong in vitroproliferative responses to recall antigens have been found to expresshigh levels of the adhesion receptors CD29 and CD44, in addition toenhanced levels of three other adhesion receptors, CD2 and its ligandLFA-3, and the leukocyte integrin LFA-1 (Sanders et al., J. Immunol.140: 1401-1407, 1988). They have also been reported to express greaterlevels of VLA-4 and VLA-6 (Shimizu et al., 1990). Memory T cells havealso been reported to exhibit enhanced in vitro adhesive behavior ascompared to T cells of naive phenotype, including enhanced adhesion toendothelial cells and homotypic adhesion (Pitzalis et al., Eur. J.Immunol. 18: 1397-1404, 1988), and, enhanced adhesion to fibronectin andlaminin upon treatment with PMA or crosslinking with anti-CD3 (Shimizuet al., 1990). Migration and recirculation of memory T cells alsoappears to be different from that of naive T cells. T lymphocytes ininflammatory infiltrates in humans have been found to be largely ofmemory phenotype (Pitzalis et al., 1988). Studies in sheep (C. R. Mackayet al., J. Exp. Med. 171 801-817, 1990) indicated that naive and memoryT cells, respectively, exhibit two distinctive pathways of recirculationbetween the circulatory and peripheral lymphatic systems. Naive T cellsappear to follow the well-known recirculation pathway from blood toperipheral lymph nodes, presumably via high endothelial venules (HEV) inthe lymph nodes. (Butcher, E. C., Curr. Top. Microbiol. Immunol., 128:85-122, 1986; Duijvestijn et al., Immunol. Today 10: 23-28, 1989).Memory T cells, however, appear to recirculate from blood intoperipheral tissues. In this connection, most, but not all peripheralblood memory T cells appear to lack the Mel 14/Leu8 homing receptor,which is at least in part involved in adhesion of lymphocytes to HEVleading to migration of the lymphocytes into peripheral lymph nodes(Camerini et al., Nature 342:78-82, 1989; Tedder et al., J. Immunol.144: 532-540, 1990). The mechanism of migration of memory T cells intoperipheral tissues and in particular into inflammatory sites is unknown.However, it is possible that VLA-4 adhesion to VCAM-1 could provide onemode of such migration.

In this connection, Goodman and Lefrancois (J. Exp. Med. 170: 1569-1581,1990) have recently shown that not only the majority murine intestinalintraepithelial T cell population bearing TCR γδ, but also the minorityTCR αβ population, possess specific phenotypic features ofintraepithelial T cells (T. Goodman and L. Lefrancois, J. Exp. Med. 170:1569-1581, 1990). These features include constitutive cytolyticactivity, having a large proportion of cells Thy1⁻, and expression on alarge proportion of cells of the leukocyte common antigen(T200)-associated carbohydrate differentiation antigen, T200. The Thy⁻and CT1⁺ phenotypes are not seen with other peripheral T cells, and mayreflect the effect of the intestinal milieu on T cell function and/orspecific "IEL" (intraepithelial lymphocyte) lineages of αβ and γδ Tcells which specifically home to the intestinal mucosa and carry outspecific functions within the milieu. If the latter is the case then thedifference between mouse and human IEL may mainly be a quantitative one,reflecting a higher proportion of TCR αβ IEL than in the case of themouse. In the case of mouse or human TCR αβ intestinal IEL, these cellscould also possess the homing receptors and other function-specificphenotypic markers of TCR γδ cells with similar localization andfunctional properties.

One type of TCR γδ T cell with specific localization properties alsoappears to possess distinctive ECM adhesion properties (Maxfield et al.,J. Exp. Med. 169: 2173-2190, 1989). Cultured lines of murine dendriticepidermal T cells (DETCs), the TCR γδ cells which specifically localizeto skin, exhibit adhesion to fibronectin, fibrinogen, and vitronectin.This binding can be blocked both by RGDS and specific monoclonalantibodies to a dimeric protein with a structure which resembles membersof the integrin family. As discussed both by Maxfield et al. and Hemler(1990), in structure and ECM specificity, this protein resembles a β₁integrin, and may be a known or new member of this family. The proteinis also expressed on murine splenic T cells very late after stimulationwith Con A (similar to VLA-1 and VLA-2); however no adhesion to ECM of Tcells other than DETCs mediated by this protein has yet been reported.Adhesion to ECM proteins of DETCs mediated by this receptor couldtherefore be involved in the trafficking and/or function of DETCs.

Isolation of Cells From IL-2 Cultured PBLs Which Adhere to Collagen IVPlates in the Presence of Laminin

Initial experiments were designed to define conditions under which afraction of IL-2 cultured PBLs would adhere to collagen IV coatedplates. In cells cultured in IL-2 containing medium for 1-2 days, suchadhesion, in laminin containing complete medium, appeared to be completewithin 2-4 h. The extent of adhesion appeared to be greater in 2 daythan in either 1 or 3 day cultures. Adhesion of day 2 cultured PBLs tocollagen IV coated plates in the presence of 10 μg/ml laminin variedfrom about 1-5% of the total cells with PBLs cultured from differentnormal donor blood samples, while adhesion to uncoated plates wasgenerally 0.5% or less. Adhesion to collagen IV coated plates in theabsence of laminin was low; 10 μg/ml laminin appeared to be optimal andwas found to increase adhesion to collagen IV plates 2-4 fold. Adhesionto laminin-coated plates (in the absence of collagen IV) or to untreatedplates in the presence of 10 μg/ml. laminin was no more than that tountreated plates in the absence of laminin.

Analysis of the Surface Marker Phenotypes of PBLs Which Are Adherent toCollagen IV/Laminin Plates

Adherent cell populations isolated from different normal donor bloodsamples were found to consist of variable proportions of CD3⁺ and CD3⁻cells (between 35-55% CD3⁺ in three experiments). T cells present inthese populations contained a high proportion of TCRδ1⁺ cells (between25-85%), as assessed by staining with the pan-TCRγδ antibody TCRδ1,described above. Although the proportion of T cells bearing TCRγδ in theadherent sub-population was high in the three experiments performed,only between 5-10% of total TCRγδ cells in the input population adheredto the plates. Between 0.5-2% of total CD3⁺ cells were found to beadherent.

Because the number of cells adhering to collagen IV/laminin plates waslow, and because populations cultured from the adherent cells were foundto consist entirely of T cells, the phenotypes of the CD3⁻ adherentcells were not characterized further. However, results from oneexperiment (data not shown) suggest that at least a large fraction ofthe CD3⁻ may be NK cells. When PBL were treated with antibodies to theNK marker NKHlA and CD16 and complement prior to culture in IL-2, thenumber of cells in the day 2 culture which adhered to collagenIV/laminin plates was reduced by a factor of about two as compared to aparallel culture derived from non-antibody treated cells. The CD3⁻ andtotal CD3⁺ fractions of adherent cells were also reduced by about afactor of two; however, the TCRδ1⁺ fraction was only reduced by about20%. This indicates that in this culture, a large fraction of both theCD3⁻ cells and the CD3⁺ cells which did not express TCRδ1 expressed oneor both of the markers Leu19 and CD16, while a smaller proportion of theTCRδ1⁺ cells did so. Note that EMATs have been demonstrated from all 6normal donors examined.

Expansion of Collagen IV/Laminin Adherent PBLs in IL-2 Containing Media

Although we have observed spontaneous long-term growth of culturesisolated from two collagen/laminin adherent cell populations isolatedfrom PBLs of normal donors (one of which is the cell line PW-EMAT), inmost cases only limited expansion is achieved without reactivation ofthe cells using PHA and irradiated feeder cells. However, once rapidgrowth is achieved, the cells were found to expand 4-5 orders ofmagnitude over as long as 4-6 weeks, without a need for further periodicreactivation. This is illustrated for example by the cell line ZB-EMAT,which required two rounds of reactivation before it gave long-term,continuous expansion.

Therapeutic Applications

ECM binding cells isolated and cultured using the present methods fromIL-2 activated PBL mixtures can be infused into cancer patients as withLAK or TIL adoptive immunotherapies. EMAT cells are expected to makelL-2 and also may require less IL-2 than with LAK cells, thus reducingthe severity of the IL-2 side-effects of treatment (Rosenberg et al., N.Engl. Jour. Med. 319:1676 (1988)). Alternatively, EMATs, as with TILsand other T cells, could be grown in low-dose IL-2 supplemented withIL-4, which is expected to result in their ability to grow afterinfusion into patients with administration of low dose, subtoxic levelsof IL-2 (Kawakami et al., Jour. Exp. Med. 168: 2183 (1988)). In theextreme case, EMAT cell IL-2 production could suffice to maintain theirviability and activation. One would then not need to infuse lL-2 intopatients thus making treatments less expensive; eliminate most majorlL-2 undesired side-effects; and even make possible outpatient treatmentin many cases. The latter also is expected to be facilitated if EMATcells are indeed more effective per unit than LAKs or TILs, therebyreducing the numbers of cells needed for treatment.

When EMAT cells are infused into patients, being T cells, they areexpected to migrate into the spleen, liver, and the lymph notes; withless trapping in the lungs and liver than with LAKs. Indeed, γδ T cellsare generally seen to reside in spleen and lymph nodes normally. It isexpected that EMAT in the lymph nodes will be able to transitendothelial cell layers and basement membranes in the local circulationand enter the surrounding tissues. In those lymph nodes which are withinthe local region of neoplastic tumors, this facilitates the entrance ofEMAT cells into the substance of the tumors. Location of tumors by EMATcells which have entered the neoplastic tissues is expected to befacilitated by the well-known phenomenon of breakdown of basementmembranes by metastatic tumors (G. Marin and R. Timpl, Annu. Rev. CellBiol. 3:57 (1987)). Cells with receptors for collagen IV and/or lamininhave been observed to respond chemotactically to gradients of breakdownproducts of these basement membrane components (T. J. Herbst et al.,Jour. Cell Biol. 106:1365 (1988)). Extensive ameboid motion by EMATcells has been observed-- a capability which would fit in and supply themotility needed for these migrations and tumor penetrations.

A second way that EMAT cells are expected to reach tumors in vivo reliesupon the phenomenon of angiogenesis (the formation of new capillaryblood vessels) in tumors. It has been observed that such tumorinfiltrating blood vessels are often disordered, and contain nakedbasement membrane proteins R. L. Carter, in Invasion: Experimental AndClinical Implications, Oxford University Press, 1984, p. 168].Circulating EMATs are expected to bind to these naked basementmembranes; transit them; and thus penetrate the tumor in vivo.

When EMATs enter tumors, they are deemed to carry out a number ofanti-tumor activities. The first is cytotoxicity or direct killing oftumor cells. Other cell activities would be dependent on lymphokinesand/or other factors such as complement components and leukotrienesbeing produced by the EMATs cells. One secondary activity would beactivation of local capillary endothelial cells. This activity isexpected to result in the appearance of new receptors on the surface ofthese endothelial cells; and then allow for the binding and transit intothe tumor of macrophages, neutrophils, and/or αβ T cells which possesshoming receptors for the activated endothelial cells involved ininflammation.

A third activity is expected to be activation (via thelymphokines--interferon, GM-CSF, and complement components) of localdendrites and of macrophages which have entered the tumor via the abovemechanism. These antigen presenting cells should then process tumorantigens released into the medium via lysis of tumor cells by EMATcells; and interact with αβ T cells having specificity for tumorantigens sufficient to activate them and induce their proliferation.This, in turn, is expected to result in the growth of new, activatedhelper and cytotoxic T lymphocytes which will carry out immune activityagainst the tumor. Increasing infiltrates of macrophages and neutrophilsbrought in both by the original EMAT cells and perhaps by the helper Tcells, being similar to a delayed hypersensitivity reaction are expectedto damage local capillaries, resulting in deprivation of oxygen andnutrients to the tumor and lead to massive destruction of the tumor.

In summary, EMAT cells are expected to reach tumors in vivo via transitof the naked basement membranes in the disordered blood vessels inducedby the tumors themselves; and/or via transit of intact endothelialvenules and/or capillaries in areas of inflammation including tumors,and/or by their own specific homing receptors and capabilities. EMATcells should then destroy tumor cells via direct cytotoxic activity; andthen cause immune initiation and amplification via both theirlymphokines and their release of tumor antigens. This also is expectedto result in amplified immune reactions mediated by other components ofthe immune system, notably classical helper and cytotoxic αβ T cells andmacrophages.

Use of EMAT and Other Extracellular Matrix Binding T Cells ExhibitingLong Term Continuous Growth in Gene Therapy

In one known "gene therapy" approach, human cancer patients have beeninfused with TIL transformed with a retroviral vector containing arecombinant drug resistance gene (neoR). Gene therapies involving using,as drug delivery systems, cells transformed with vectors coding forvarious therapeutic proteins, have been used or suggested for use intreating various conditions, including genetic deficiency diseases (suchas ADA deficiency) and other conditions including ones that are notdirectly caused by mutations, such as, for example, cancer andemphysema. It has been proposed to use such a transformed TIL-based genetherapy approach for treating children with adenine deaminase (ADA)deficiency by infusing each patient with a preparation containing Tcells that were isolated from the patient and transformed with a vectorcarrying a gene for the ADA enzyme.

An ideal cell for systemic gene therapy is a bone marrow stem cell, butthese are difficult to isolate and cultivate, and one known approach fortreatment of cancer patients uses TILs. An ideal cell for gene therapyof a local condition (including such non-cancer conditions as emphysema,for which the enzyme α1 anti-trypsin is believed to be a therapy), is acell that can localize to the affected area.

TILs or other mixed T cells are difficult to grow and requirereactivation, and for these and other reasons they may not be an ideal Tcell to use for gene therapy. EMATs or other extracellular matrix (ECM)adherent T cells, according to the invention, especially those whichexhibit long term continuous growth, can be preferable for genetherapies for a number of reasons. They are easier to cultivate. Theycan localize and distribute more widely (such as, for example, in lymphnodes). They can be longer-lived, because 1) unlike TILs or activatedPBL which are largely trapped in liver where they are expected to bemostly destroyed, EMATs and other ECM adherent T cells, if distributedto lymph nodes or other tissue sites, would not be destroyed, and 2) inthe case of γδ EMATs, studies in the mouse suggest that γδ T cells maybe longer lived (at least on the average) than αβ T cells, and 3) atleast many of populations of ECM adherent T cells from a particularsource site, these can be expected upon return to the source animal tolocalize at the source site. Such a treatment using substantiallypurified ECM adherent cells according to the invention can be used, forexample, to localize gene therapy for emphysema.

For use of gene therapies in adoptive immunotherapies for cancer,adhesion-capable T cells, and especially EMATs, can be used forsite-directed delivery of, for example, lymphokines (e.g., IL-2, IL-4,GM-CSF), by exploiting their capacity for localization to a site and forcontrolling, via binding to extracellular matrix protein at the site,expression and production at the site of lymphokines to amplify otherimmune cells and endothelial cells. Adhesion-capable cells according tothe invention that are capable of localizing to a site can betransformed to ensure that they are capable of producing the lymphokinesat the site. It has been suggested that binding of CD4 helper T cells(largely memory T cells) can result in expression of the IL-2 gene bythe cells. It has also been suggested that binding of murine DETC (γδskin T lymphocytes) to fibronectin can result in secretion of GM-CSF orof IL-4 (depending on which DETC clone is used.)

A control region for extracellular matrix binding induction of geneexpression in T cells can be used in an expression system to transforman ECM binding T cell to effect ECM binding-mediated induction oflymphokine production by the T cell only at a selected site containingthe selected ECM protein. This can provide a particular advantage wherethe therapeutic substance being used can be lethal to the patient ifexpressed systemically (such as, for example, tumor necrosis factor,"TNF"), or can have widespread side effects (as, for example, IL-2).Moreover, this control of expression of genes which is dependent onlocalization and extracellular matrix binding may also be useful intreatment of emphysema (as described above) or other use of gene therapyfor treatment of a condition in a local site.

I claim:
 1. Substantially purified normal, mature T cells capable ofbinding in the absence of serum to a collagen, wherein said T cell bindsto collagen IV>collagen I>fibronectin, and not to laminin or fibrinogen.2. A method for increasing the proportion, in a cell population, ofnormal, mature T cells capable of binding to an extracellular matrixcomponent, comprising steps ofproviding a cell mixture comprisingactivated T lymphocytes in a medium, contacting the activated Tlymphocytes with a collagen on a support in the presence of solublelaminin to permit at least a portion of the activated T lymphocytes toadhere to said collagen on said support, and separating said medium fromsaid support together with any cells in said medium not adhering to saidcollagen on said support, and retaining the portion of activated Tlymphocytes that adheres to said collagen.
 3. The method of claim 2wherein the step of providing a cell mixture comprising activated Tlymphocytes comprises steps ofcontacting a cell mixture comprisingmononuclear leukocytes including T lymphocytes with a plastic supportsurface to permit monocytes and macrophages present in the cell mixtureto adhere to said plastic support surface, and separating said plasticsupport surface and any cells adhering thereto from the cell mixturecomprising T lymphocytes.
 4. The method of claim 2 wherein the step ofproviding a cell mixture comprising activated T lymphocytes comprisessteps of providing a cell mixture comprising T lymphocytes,andactivating cells in the cell mixture to produce the cell mixturecomprising activated T lymphocytes.
 5. The method of claim 3 wherein thestep of providing a cell mixture comprising activated T lymphocytesfurther comprises the step ofactivating cells in the cell mixture toproduce the cell mixture comprising activated T lymphocytes.
 6. Themethod of claim 4 or 5 wherein the cell mixture comprising T lymphocytescomprises cells derived from a first source animal, and said activatingstep comprises contacting the cell mixture comprising T lymphocytes withlymphocytes derived from a second source animal.
 7. The method of claim6 wherein said lymphocytes derived from said second source animalcomprise irradiated lymphocytes.
 8. The method of claim 4 or 5 whereinsaid activating step comprises contacting the cell mixture comprising Tlymphocytes with a lectin and with irradiated peripheral bloodmononuclear cells.
 9. The method of claim 8 wherein said irradiatedperipheral blood mononucleocytes and at least a portion of the cellmixture comprising T lymphocytes are derived from the same sourceanimal.
 10. The method of claim 8 wherein at least a portion of the cellmixture comprising T lymphocytes is derived from a first source animal,and said irradiated peripheral blood mononuclear cells are derived froma second source animal.
 11. The method of claim 4 or 5 wherein saidactivating step comprises contacting the cell mixture comprising Tlymphocytes with a mitogenic antibody.
 12. The method of claim 11wherein said mitogenic antibody comprises a monoclonal antibody.
 13. Themethod of claim 12 wherein said monoclonal antibody is selected from thegroup consisting of anti-CD2, anti-CD3, and anti-T cell receptormonoclonal antibodies.
 14. The method of claim 4 or 5 wherein saidactivating step comprises contacting the cell mixture comprising Tlymphocytes with an extracellular matrix component different incomposition from said collagen on said support.
 15. The method of claim4 or 5 wherein said activating step comprises contacting the cellmixture comprising T lymphocytes with mixed B lymphoblastoid cellstogether with a combination of compositions selected from the groupconsisting of irradiated heterologous peripheral blood lymphocytes, alectin, a mitogenic antibody and an extracellular matrix protein boundto a support.
 16. The method of claim 4 or 5 wherein said activatingstep comprises contacting the cell mixture comprising T lymphocytes witha first lymphokine-containing medium.
 17. The method of claim 16 whereinsaid lymphokine in said first lymphokine-containing medium comprises aninterleukin.
 18. The method of claim 17 wherein said lymphokine in saidfirst lymphokine-containing medium comprises interleukin-2.
 19. Themethod of claim 17 wherein said lymphokine in said firstlymphokine-containing medium comprises interleukin-4.
 20. The method ofclaim 16 wherein said lymphokine in said first lymphokine-containingmedium is selected from the group consisting of tumor necrosis factor,granulocyte macrophage colony stimulating factor, and γ interferon. 21.A method for assessing the likelihood that a mixture of cells containsactivated, normal, mature T cells capable of localizing to a site invivo, wherein an extracellular matrix component is present at the site,comprisingcontacting said mixture of cells with a collagen on a supportin the presence of soluble laminin, under conditions that permit bindingto said collagen on said support of T cells that are capable of bindingto the collagen,whereby greater binding of T cells to said collagen onsaid support indicates a greater likelihood that said mixture of cellscontains activated T cells capable of localizing to the site in vivo.22. A collagen binding, normal T cell capable of long term continuousgrowth.